[CANCER RESEARCH 52, 6216-6223, November 15. 1992]
Intra- and Interindividual Variability in Systemic Exposure in Humans to
2-Amino-3,8-dimethylimidazo[4,5-/]quinoxaline and 2-Amino-l -methyl6-phenylimidazo[4,5-o]pyridine, Carcinogens Present in Cooked Beef1
Anthony M. Lynch,2 Mark G. Knize, Alan R. Boobis, Nigel J. Gooderham, Donald S. Davies, and
Stephen Murray
Department of Clinical Pharmacology, Royal Postgraduate Medical School, Ducane Road, London Wll ONN, United Kingdom [A. M. L., A. R. B., N. J. G.,
D. S. £>.,
S. M.], and Biomédicaland Environmental Sciences Divisions, Lawrence Livermore National Laboratory, Livermore, California 94550 ¡M.G. K.j
ABSTRACT
idazopyridine families (4, 5). The most abundant members of
these families (Fig. 1) include MelQx,3 DiMelQx, and PhIP.
During the cooking of beef, the genotoxic heterocyclic aromatic
amines 2-amino-3,8-dimethylimidazo[4,5-/]quinoxaline
(MelQx),
2amino-3,4,8-trimethylimidazo[4,5-/]quinoxaline
(DiMelQx),
and 2amino-l-methyl-6-phenylimidazo[4,5-6]pyridine
(PhIP) are formed. Little
MelQx and DiMelQx are among the most potent bacterial
mutagens discovered and are present at levels of 1-3 ng/g in
cooked meat (4, 6). However, these compounds are at best only
weakly positive in mammalian genotoxicity test systems (7-10).
In contrast, although PhIP is a much less potent bacterial mutagen, it is present in meat at levels several times higher than
MelQx and DiMelQx (4) and is far more effective at inducing
DNA damage in mammalian cells than the other two com
pounds (11-13). In rodent bioassays, MelQx and PhIP are
carcinogenic, inducing tumors in a variety of tissues (14-18),
including, in the case of PhIP, the colon and mammary glands
(19). Recently, 2-amino-3-methylimidazo[4,5-/)quinoline
(an
aminoazaarene structurally related to MelQx and also found in
cooked meat) has been shown to be a potent hepatocarcinogen
in nonhuman primates (20).
Heterocyclic amines require metabolic activation to their ul
timate mutagenic/carcinogenic
species. The metabolism of
MelQx and PhIP has been studied extensively in vitro (21-26)
and in vivo (27-32). In rodents, the principal pathways of
biotransformation have been elucidated. Both compounds are
activated by A'-oxidation which is catalyzed by isoenzymes of
the P-450-dependent mixed function oxidase system [the initial
activation of MelQx and DiMelQx is catalyzed primarily by
CYP1A2, whereas PhIP activation also involves CYP1A1 (33)].
The resulting TV-hydroxy arylamines, the proximate carcino
gens (34), may be further metabolized to highly reactive A'-sulfonyloxy, ./V-proIyloxy, or jV-acetoxy esters which can covalently
bind to DNA and may initiate carcinogenesis (35).
The major pathways of MelQx and PhIP metabolism involve
cytochrome P-450-mediated hydroxylation at the C-5 position
of MelQx and at the C-4 position of the phenyl ring of PhIP,
followed principally by conjugation to sulfuric and/or glucuronic acid, although minor pathways include conjugation with
glutathione and acetic acid (36-39). Phase II reactions involv
ing the hydroxylated exocyclic amine group have also been
reported and lead to the formation of metastable conjugates
which can be hydrolyzed enzymatically, or by acid, back to the
parent ./V-hydroxy arylamine (26). In rodents, imidazoquinolines and imidazoquinoxalines
are extensively metabolized,
with the products excreted more in the feces than in the urine.
In the rat, MelQx is almost completely absorbed and at low
doses is almost completely metabolized, with 45% of the dose
is known about the fate of these compounds in humans or the factors
affecting it. We have developed assays based on capillary column gas
chromatography-negative
ion mass spectrometry capable of the simulta
neous measurement of MelQx, DiMelQx,
human urine using stable isotope labeled
male volunteers were invited to consume
(400-450 g lean beef, cooked as patties
and PhIP in cooked meat and in
analogues. Ten normal, healthy
a standard cooked meat meal
on a griddle hotplate) on four
separate occasions over a period of 14 months. Following consumption of
the test meals, urine was collected from 0 to 8 h, during which time all free
amines were excreted and analyzed for MelQx, DiMelQx, and PhIP.
Subjects ingested 240 ±9 (SEM) g cooked meat, which contained 2.2 ±0.2
ng MeIQx/g meat, 0.7 ±0.1 ng DiMeIQx/g meat, and 16.4 ±2.1 ng
PhIP/g meat. The variability in relative systemic bioavailability was
assessed from the percentage of ingested amine excreted unchanged in the
urine. Subjects excreted 2.1 ±1.1% of MelQx and 1.1 ±0.5% of PhIP
ingested as unchanged amine in the urine. Levels of DiMelQx in urine, if
present, were below the sensitivity of our assay (20 pg/ml) and could not be
detected in any of the samples analyzed. Irrespective of dose, urinary ex
cretion of unchanged MelQx or PhIP (expressed as a percentage of the
ingested dose) remained constant for each individual subject. The intraindividual coefficients of variation for MelQx (28.4%) and PhIP (23.7%)
were low and the pooled interday (intrasubject) coefficients of variation for
both compounds were only 19 and 3.4%, respectively. In contrast, intersubject (intraday) variation was greater, with pooled coefficients of varia
tion of 145% for MelQx and 71% for PhIP. Based on these studies, it
should be possible to use the percentage excretion of MelQx and PhIP to
assess the relative bioavailability of these compounds in humans.
INTRODUCTION
A substantial proportion of all human cancer is associated
with diet (1, 2). The normal cooking and processing of meatcontaining food generates a number of chemicals which are
positive in bacterial mutagenicity assays (3). Among these are
several heterocyclic amines of the imidazoquinoxaline and im-
Received 4/27/92; accepted 9/11/92.
The costs of publication of this article were defrayed in part by the payment of
page charges. This article must therefore be hereby marked advertisement in accord
ance with 18 U.S.C. Section 1734 solely to indicate this fact.
1 Work carried out at the Royal Postgraduate Medical School was supported by
grants awarded by the National Cancer Institute, Department of Health and Hu
man Services (USPHS Grant CA40895-02), Ministry of Agriculture, Fisheries and
Food, and the Cancer Research Campaign. Work at the Lawrence Livermore
National Laboratory was performed under the auspices of the United States DOE
under contract W-7405-ENG-48 and supported by National Institute for Environ
mental Health Sciences Agreement 22YO1-ES-10063 and National Cancer Insti
tute Grant ROI-CA40811.
2 To whom requests for reprints should be addressed.
•¿'
The abbreviations used are: MelQx. 2-amino-3,8-dimethylimidazo[4,5-/]quinoxaline; DiMelQx. 2-amino-3,4,8-trimethylimidazo[4,5-/|quinoxaline;
PhIP,
2-amino-l-methyI-6-phenylimidazo[4.5-Alpyridine;
[13C.15N2]MeIQx.2-[15N]amino-3,8-dimethyl[l-15N,2-13C]imidazo[4,5-/]quinoxaline;
[2H5]PhIP.2-amino-lmethyl-6-pentadeuterophenylimidazo[4,5-A]pyridine; -bisTFMB, -bistrifluoro-methylbenzyl bromide; CV, coefficients of variation; ANOVA, analysis of variance.
6216
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HUMAN EXPOSURE
TO CARCINOGENS
PRESENT
IN COOKED BEEF
PhIP in a group of subjects ingesting these compounds by the
consumption of a cooked beef meal on several separate occa
sions.
MATERIALS
AND METHODS
Chemicals
CH,
Fig. 1. Chemical structures of MelQx (<j), DiMelQx (6), and PhIP (c).
being excreted in the urine but with less than 2% as the un
changed compound. At higher doses, there is evidence of con
siderable saturation of some of the metabolic pathways; the
percentage of the dose excreted in the urine decreases to 20% at
20 mg/kg, almost all of which is unchanged compound (40).
However, following induction of P-450 the excretion profile
resembles that obtained with the lowest doses, indicating unsaturation of oxidative metabolism. PhIP is incompletely ab
sorbed in the rat, with approximately 40-50% of a p.o. dose
remaining unabsorbed. Only small amounts of PhIP are ex
creted unchanged in the urine, 1.5-3%, depending upon the
dose, with a total of 15-25% of the dose eliminated by the
kidneys (41). Thus, in rodents the extent of MelQx and, to a
lesser extent, PhIP metabolism (and as a consequence the
amount of unchanged amine excreted in the urine) depend upon
both the size of the dose administered and the degree of P-450
induction (40, 41).
Daily human exposure to these heterocyclic amines is con
siderably less than the doses used in animal carcinogenicity
studies. Human liver can readily ¿V-hydroxylateheterocyclic
amines, while both human liver and colon are able to catalyze
their O-acetylation to proximate genotoxic metabolites (42). It
has recently been shown that the /V-hydroxylation step is cata
lyzed almost entirely by CYP1A2, at least in the liver, and that
it accounts for >88% of the oxidative metabolism of MelQx
(42-46) and >90% of the oxidative metabolism of PhIP (4647). Hence, the relative risk posed to humans by heterocyclic
amines, determined in rodent carcinogenicity studies, is likely
to be underestimated because of reduced metabolism relative to
dose. It is therefore important to determine the fate of these
compounds in humans at the doses encountered on a normal
diet.
These results, together with the potential risk posed to hu
man health by heterocyclic amine exposure in the diet, have
prompted the need to determine the fate of these compounds in
human beings. We have previously reported an assay for
MelQx and DiMelQx in fried beef using gas chromatographynegative ion mass spectrometry (6). This was subsequently
modified to allow the detection and measurement of MelQx in
human urine after consumption of a cooked meat meal (48).
This methodology has now been extended to include the anal
ysis of PhIP in meat and urine and all three amines can be
measured in a single Chromatographie run. The new assay has
been used to assess the relative bioavailability of MelQx and
MelQx, DiMelQx, and PhIP were obtained from Toronto Research
Chemicals Inc. (Downsview, Ontario, Canada). Stock solutions of the
three compounds in methanol of varying dilution were prepared and
stored at -20°C until required. Chemicals used in the synthesis of
[2H5]PhIP were purchased from Aldrich Chemical Co. Ltd. (Milwau
kee, WI). 3,5-Bistrifluoromethylbenzyl
bromide was supplied by
Fluorochem, Ltd. (Glossop, United Kingdom) while diisopropylethylamine and dodecane were obtained from Sigma Chemical Co. Ltd.
(Poole, United Kingdom). Acetonitrile, ethyl acetate, methanol, dichloromethane, and hexane were all of Anular grade and acetonitrile and
ethyl acetate were redistilled before use.
Protocol
Ten normal, healthy male adult volunteers were invited to consume
a standard cooked beef meal on 4 separate occasions over a period of
14 months (an 11th person dropped out of the trial after the first study
day, see Table 1 for demographic data). Local Ethical Committee ap
proval and written informed consent from each volunteer were obtained
before the study commenced. Subjects were requested not to eat meat or
any meat products on the day before the study (there were no other
dietary restrictions) and on the study day to fast until the test meat meal
was consumed. Lean rump beefsteak (~400-450 g/person) was pur
chased locally, coarsely minced, and molded into 4 equal-sized patties
(diameter, ~9 cm; depth, 2 cm). The meat was cooked on a griddle hot
plate (without added fat or oil) for 10-15 min at 200-250T until well
browned. Before consuming the meat, subjects were requested to void
their bladders and to collect a control sample of urine (~50 ml). Once
cooked, a portion of each patty was removed and stored at —¿
20°Cfor
subsequent analysis, and the remaining meat was weighed. The subjects
then consumed the cooked meat together with tap water (~400 ml).
After completing the test meal subjects were requested not to consume
further food or drink for 6 h. On the first leg of the study, urine was
collected for the next 36 h (0-4 h, 4-8 h, 8-12 h, 12-24 h, 24-36 h
collections) to determine the elimination of MelQx, DiMelQx, and
PhIP. In subsequent parts of the study, urine was collected for the first
8 h after ingestion of meat. The volumes of the urine collections were
measured and urine pH and normality were determined with N-Multistix S. G. diagnostic strips (Ames Division, Miles Laboratories, Ltd.,
Slough, United Kingdom). Samples (—¿
50 ml) from each urine collec
tion were stored at -20°C until analysis.
Table 1 Human demographic data
(no. of meat
meals/week)0-38-120-313-208-124-74-74-78-124-74-7Smokin
Subject\"2345678»910IIeAge
(yr)4226283125353623263037Diet
(cigarettes/day)5-10<1<1
" The normal weekly meat diet of subject 1 was restricted to fish.
* Subject 8 gave up smoking (~5 cigarettes/day) several months before the trial
commenced.
c Subject 11 was present for the first study day only.
6217
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HUMAN EXPOSURE
TO CARCINOGENS
PRESENT
IN COOKED BEEF
Standards
Synthesis
|'-'C,l5N2|MeIQx.
The synthesis of this compound has been de
scribed elsewhere (6).
|2H5|PhIP. [2H5]PhIP was prepared from 5-amino-2-chloropyridine
by a four-step synthetic route (Fig. 2). Full details of this synthesis will
be published elsewhere.
For the analysis of amines in cooked meat, six standards containing
[l3C,15N2]MeIQx (5 ng), [2H5]PhIP (25 ng), and various amounts of
MelQx (0-4 ng), DiMelQx (0-4 ng), and PhIP (0-20 ng) in methanol
(150 M!)were prepared. For urine analysis, six standards containing
one-half of these quantities of internal standards and amines were pre
pared. These solutions were stored at —¿20°C
prior to derivatization.
Extraction of Cooked Meat
The portions of the cooked patties which had been stored at -20°C
Derivatization Procedure
for amine analysis were allowed to thaw at room temperature. They
were then coarsely minced in a domestic food processor. [IJC,ISN2]MelQx (10 ng) and [2H5]PhIP (50 ng) in methanol (200 and 100 /il,
respectively) were added to a sample of the chopped meat (2 g), and the
mixture then homogenized in 0.25 N HC1 (10 ml) with a Polytron tissue
homogenizer (Kinematica GmbH, Lucerne, Switzerland). After centrifugation, an aliquot of the acidic aqueous supernatant (5 ml) was trans
ferred to a clean glass tube and washed with dichloromethane (5 ml)
by manual inversion and then separated by centrifugation. The upper
aqueous layer was transferred to a clean tube while the lower organic
layer was discarded. This washing procedure was repeated twice more,
then l M sodium carbonate solution (3 ml) was added to the aqueous
phase and the alkaline product was extracted with ethyl acetate (2x5
ml). The combined organic extract was evaporated to dryness under
nitrogen and the residue was transferred to a half-dram glass vial
with methanol (2 x 0.75 ml). Samples were stored at -20°C prior to
Methanol present in standards, meat, and urine extracts was re
moved by evaporation under nitrogen. A 5% solution of 3,5-bistrifluoromethylbenzyl bromide in acetonitrile (80 n\) and diisopropylethylamine (20 n\) were added to each vial, and the reaction mixture was left
at room temperature overnight and then evaporated to dryness under
nitrogen. To the residue were added 0.1 N HC1 (200 ^1) and hexane
(750 M!)-The vial contents were vortex-mixed and centrifuged and the
upper organic layer was discarded. This washing procedure with hexane
was repeated: then l Nsodium carbonate solution ( 100 M!)was added to
the aqueous phase and the alkaline product was extracted with ethyl
acetate (2 x 750 ¿il).
The combined organic extract in a half-dram glass
vial was evaporated to dryness under nitrogen and the residue was
reconstituted in dodecane (50 /il for standards and 20 /»I
for meat and
urine extracts). Aliquots of 2 n\ were injected into the gas chromatograph-mass spectrometer.
derivatization.
Gas Chromatography-Mass Spectrometry
Extraction of Urine
Internal standards, [I3C,15N2]MeIQx (2.5 ng) and [2H5]PhIP (12.5
ng) in methanol (50 and 25 ßl,
respectively) were added to urine samples
(5 ml) which were then mixed by manual inversion. Sodium carbonate
solution (1 M;2.5 ml) was added and the alkaline product extracted with
ethyl acetate (2x10 ml). The combined organic extract was transferred
to a clean glass test tube together with 0.1 N HC1 (1 ml). The tube
contents were mixed by manual inversion and separated by centrifuga
tion, and then the upper organic layer was discarded. The aqueous
phase was washed with ethyl acetate (10 ml) and then evaporated to
dryness under nitrogen, and the residue was transferred to a half-dram
glass vial with methanol (2 x 0.75 ml). Samples were stored at -20°C
prior to derivatization.
A Finnigan-MAT 4500 combined gas chromatograph-quadrupole
mass spectrometer system (Finnigan-MAT, San Jose, CA) was used.
The gas Chromatograph was equipped with a 15-m DBS J&W fused
silica capillary column which was routed through the separator oven
(maintained at 290°C)and directly into the mass spectrometer ion
source. Helium was used as carrier gas at a head pressure of 10 psi. The
gas Chromatograph was fitted with a Grob-type capillary injector oper
ated in the splitless mode and maintained at 270°C.The gas Chroma
tograph oven temperature was held at 200°Cfor 1 min, then raised to
320°Cat 20°Cmin-', and held at 320"C for 1 min. Under these con
ditions, the retention time of the di-bisTFMB derivatives of MelQx and
[uC,l5N2]MeIQx was 5.65 min; of DiMelQx the retention time was
5.90 min, and of PhIP and [2H5]PhIP it was 6.65 min. The mass
spectrometer was operated in the negative ion chemical ionization
mode with an electron energy of 100 eV. Ammonia gas was admitted to
an indicated ion source pressure of 0.4 T and the indicated ion source
temperature was maintained at 150°C.The mass spectrometer was
tuned to monitor negative ions at m/z 438, m/z 441, m/z 449, m/z 452,
and m/z 454 and data acquisition and reduction were performed by an
INCOS data system using IDOS 2 software.
KOCOCH,
C(H1IN01/C,'H|
NH,
Expression of Results and Statistical Analysis
The amounts of amine ingested and excreted by each individual were
determined from the gas chromatography-mass spectrometry results
obtained for the meat and urine samples taken for analysis, and then
individual urinary amine excretion was expressed as a percentage of the
NH2
estimated ingested doses of amines for that individual. When meat
samples contained low levels of amine (MelQx < 0.5 ng/g meat; PhIP
-CH,
< 2.5 ng/g meat) as a result of undercooking, then these and the
respective urine samples were rejected from the study. The concentra
III
a) CuSC^/CHjNH,
tions (ng/g meat) of MelQx, DiMelQx, and PhIP present in cooked
meat and the percentage amounts (relative to the estimated dose) of
b) CNBr /H3PO4
°HS
unchanged MelQx and PhIP in urine were compared according to
subject and study day, using analysis of variance (2-way ANOVA with
multiple range analysis using Bonferroni correction for 95% confidence
IV
limits). A comparison of the variation in the day-to-day ratios of
MeIQx:DiMeIQx:PhIP
present in meat was performed by KruskalFig. 2. Synthesis of [2H5]PhlP. A four-step synthetic route was used to prepare
[2H5]PhIP (V) from 5-amino-2-chloropyridine (/). The chemical intermediates
Wallis one-way nonparametric analysis by ranks. Furthermore, the in2-chloro-5-pentadeuterophenylpyridine
(II), 2-amino-5-pemadeuterophenylpyritersubject and interday coefficients of variation were calculated together
dine (///), and 2-amino-3-bromo-5-pentadeuterophenylpyridine
(IV) and the final
with the pooled intrasubject and intraday coefficients of variation.
product (J7) were all isolated by flash chromatography and characterized by mass
Pooled coefficients of variation were determined by expressing the
spectrometry.
6218
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HUMAN EXPOSURE
TO CARCINOGENS
PRESENT
IN COOKED BEEF
0.95-
0.78-,
m/ z
438
m/z
449
Fig. 3. Selected ion monitoring traces for
the analysis of MelQx and DiMelQx (a) and
PhIP (*) in fried beef. Retention times of
the di-bisTFMB derivatives: MelQx (m/z 438)
and [13C,'5N2]MeIQx (m/z 441), 5.65 min;
DiMelQx (m/z 452), 5.90 min; and PhIP (m/z
449) and [2H5lPhIP (m/z 454), 6.65 min.
A
n/z
441
A
1.80-1
0.31-,
m/z
454
m/z
452
AA
5.50
5.75
6.00
retention
6.25
6.50
6.75
time (min)
mean square for the factor in question (subject or day), i.e., the pooled
small volumes of methanol were evaporated to dryness, derivatized, and then analyzed by gas chromatography-mass
spectrometry with selected ion monitoring of ions m/z 449 and 454.
Over this concentration range, the unextracted standard curve
for PhIP was linear (m = 0.026; r = 0.998) with an intercept on
the ordinate close to, and not significantly different from, the
origin (c = 0.003).
The extraction procedure previously developed for the mea
surement of MelQx and DiMelQx in cooked meat (6) was then
applied to the analysis of PhIP in fried beef. All three amines
RESULTS AND DISCUSSION
were found in cooked meat and, because of the similar retention
The methods developed in this laboratory for the analysis of times of the derivatives, could be conveniently measured in a
MelQx and DiMelQx in meat and urine (6, 48) utilize solvent
single Chromatographie run (Fig. 3). Standards identical to
extraction in conjunction with manipulation of pH. PhIP has those used for the preparation of the unextracted standard
the same aminoimidazole ring system that is present in MelQx
curve for PhIP were extracted from fried beef. The slope of the
and DiMelQx (Fig. 1) and so should extract and derivatize in extracted standard curve obtained was not significantly differ
the same way as the other two amines. Using the same extrac
ent from that of the unextracted one, indicating that PhIP and
tion and derivatization procedures for the three compounds
[2H5]PhIP had the same recovery through the extraction pro
would have the advantage of allowing their combined assay, in cedure; thus routine analysis of the amine was made by refer
that all three amines could be analyzed in a single Chromato
graphie run. However, while it is possible to use [13C,I5N2]- ence to an unextracted standard curve covering a suitable con
centration range. Recoveries of MelQx and PhIP through the
MelQx as a common internal standard for the measurement of
extraction procedure, assessed by comparison of internal stan
MelQx and DiMelQx (6), initial experiments showed that this
dard peak areas in extracted samples with those in unextracted
compound was not a suitable internal standard for the analysis
standards, were ~40 and ~30%, respectively. We have previ
of PhIP. Consequently an analogue of PhIP labeled with five
ously analyzed raw beef and have shown that there is no MelQx
deuterium atoms ([2H5]PhIP) was synthesized (Fig. 2).
or DiMelQx present (6). When this analysis was extended to
PhIP and [2H5]PhIP formed di-bisTFMB derivatives under
include PhIP, the latter compound was also found to be absent,
the experimental conditions described above and the negative
thus
confirming that MelQx, DiMelQx, and PhIP detected in
ion mass spectra of these derivatives were analogous to those of
the di-bisTFMB derivatives of MelQx and DiMelQx (6). The fried beef are produced during the cooking process.
The extraction procedure used when MelQx was first shown
mass spectra of the PhIP and [2Hs]PhIP derivatives contained
to
be present in the urine of a subject following a cooked meat
low intensity (M-l) ions at m/z 675 and 680, respectively,
meal
(48) was reexamined to determine whether it was suitable
which constituted less than 1% of the total ion current. The
most abundant ions in the mass spectra were m/z 449 and 454 for the development of a combined assay for the three amines in
urine. Experiments with blank urine to which had been added
and corresponded to loss of a bisTFMB group from the respec
tive molecular ions. The di-bisTFMB derivatives of PhIP and known amounts of the amines showed that, just as for meat
[2H5]PhIP afforded the same good limits of detection for the samples, it was possible to measure the three compounds in one
parent compounds as had been obtained for MelQx and Chromatographie run. (Recovery of the amines through the ex
traction procedure, assessed by comparison of internal standard
DiMelQx. When the mass spectrometer was set to monitor
ions m/z 449 and 454, amounts of derivative equivalent to 1 pg peak areas in extracted samples with those in unextracted stan
dards, was ~30%). However, limits of detection of the three
of the parent amine could be detected.
A standard curve for the analysis of PhIP was prepared. Six amines in urine (4 pg/ml MelQx, 20 pg/ml DiMelQx, and 10
solutions containing PhIP (0-20 ng) and [2H5]PhIP (25 ng) in pg/ml PhIP) were not the same. Consequently, while the assay
6219
SD, as a percentage of the average of the mean values for the subjects
or study days, as appropriate. The pooled coefficient of variation thus
reflects the average variance for the respective factor averaged for the
other factor. Finally, linear regression analysis was performed on the
total amounts of MelQx and PhIP ingested by each subject against the
total amounts of unchanged MelQx and PhIP present in their urine for
each study day. Statistical analysis was performed using Statgraphics
(version 5) statistical software package (STSC, Inc., Rockville, MD).
Downloaded from cancerres.aacrjournals.org on June 15, 2017. © 1992 American Association for Cancer Research.
HUMAN EXPOSURE
TO CARCINOGENS
IN COOKED BEEF
meat samples was 2.2 ±0.2 ng/g for MelQx, 0.7 ±0.1 ng/g for
DiMelQx and 16.4 ±2.1 ng/g for PhIP (n = 33 test meals).
Thus, under the cooking conditions used here, there was a mean
MeIQx:DiMeIQx:PhIP
ratio of 3:1:24. Although there was
considerable variation in the absolute amounts of amine present
in meat cooked on individual study days (Fig. 4¿>),
there was no
significant variation in the ratios of MelQx to DiMelQx ac
cording to study day. However, there was significant interday
variation in the ratios of the imidazopyridine, PhIP, to both
imidazoquinoxalines, MelQx and DiMelQx (P < 0.002, deter
mined by Kruskal-Wallis one-way nonparametric analysis by
ranks). The reasons for this are unclear but presumably reflect
the nature of the chemical synthesis of these compounds in
meat during the cooking process. It was also noted that average
(ng/g)
40-1 (A)
30 20 10 -
5 111
(/>
•¿H
o
m
PRESENT
Table 2 Urinary elimination of MelQx and PhIP ¡nvolunteers following
a cooked beef meal"
1
23456789
10
sample)<0.020"
sample)<0.050'
0-88-12Control
0.048
<0.020<0.020
0.212
<0.050ND''<0.050
0-8
8-12Control
0.040
<0.020<0.020
0-8
8-12Control
0.059
<0.020<0.020
0.173
O.050<0.050
0-8
8-12Control
0.071
<0.020ND<0.020
<0.050
<0.050<0.050
time
(IDControl
Subject467891011Collection
o
o
U
.
o
O
0-8
8-12Control
0-8
8-12Control
Day
Fig. 4. Amount of PhIP (D), McIQx (•).and DiMelQx (Ü)present in cooked
beef patties. Variation in patty amine content according to subject (A) and vari
ation in patty amine content according to study day (B). Results are ng amine/g
cooked beef; bars, SEM.
was sufficiently sensitive to measure MelQx and PhIP in hu
man urine collected after consumption of a fried beef meal,
DiMelQx could not be detected in any of the urine samples
obtained from the subjects taking part in this study. The preci
sion of the assay for MelQx and PhIP in urine was determined
by analyzing six 5-ml aliquots of a urine collection from one
subject after consumption of a fried beef meal [22 ±0.7 (SD)
pg/ml MelQx, i.e., coefficient of variation, 3.2%; 65 ±6.4
pg/ml PhIP, i.e., coefficient of variation, 9.8%], while the interday coefficient of variation for analysis by gas chromatography-mass spectrometry was <10%.
Ten normal, healthy male volunteers were invited to consume
a standard cooked meat meal [240 ±9 (SEM) g cooked weight]
on 4 separate occasions over a period of 14 months. Fig. 4a
shows the amounts of MelQx, DiMelQx, and PhIP in the
cooked meat (ng/g; mean ±SEM) estimated to have been con
sumed by each individual on the different study days. There was
no significant intraindividual variation in the amounts of amine
ingested by any individual as determined by ANOVA with a
multiple range test using the Bonferroni method (95% confi
dence limit). The average amount of the amines present in all
0.288
<0.050<0.050
0.059
0.020<0.020
0.980
<0.050<0.050
0-8
0.036
0.480
8-12MelQx(ng/5-ml <0.020PhIP(ng/5-ml
•¿C0.050
" No McIQx or PhIP was detected in any sample from 12 to 36 h or ¡ncontrol
urine samples. Subjects 1. 2. 3, and 5 received insufficiently cooked meat for
subsequent urine analysis.
* Limit of detection of gaschromalography mass spectrometry assay for MelQx.
' Limit of detection for PhIP.
rf ND. not determined because of interference in chromatography.
Table 3 Detection of MelQx and PhIP in human urine (0-8-h collection)
following a fried heef meal"
(% of dose)
Subject12345678910Amount
of MelQx1.4
urinary
(3)1.6
±0.5
(3)4.3
±0.3
(3)1.2
±1.0
(3)1.7
+ 0.1
(3)1.±0.6
(4)1.7
5 ±0.3
(4)2.1
±0.7
(3)3.7(5.4,
±1.1
(2)»1.2.0)
(4)2.1
5 ±0.3
(% of dose)
ofPhIP0.6
urinary
(3)0.9 0.2
(3)2.3 0.3
(3)0.8 0.8
(3)0.7 0.2
(3)0.8(0.9.
0.2
(2)*1.5(0.9.
0.6)
(2)*1.0(1.0.
2.0)
(2)"0.9 1.0)
(3)1.1±0.2
(2)*1.1
(1.0, 1.2)
±l.lcAmount
±0.5CMelQx/PhlP2.31.81.91.52.41.91.12.14.11.42.0
±0.8e
" Numbers in parentheses, of samples analyzed.
* Results are means ±SD. except for subject 9 for MelQx and subjects 6, 7, 8,
and 10 for PhIP in whom only 2 results could be obtained because of assay inter
ference on the other two test days. The mean and individual values are shown for
these subjects. MelQx and PhIP could not be detected in control urine samples
from any subject.
'Mean ±SD; n = 10.
6220
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HUMAN EXPOSURE TO CARCINOGENS PRESENT IN COOKED BEEF
amounts of the amines present in meat cooked from frozen
were only one-half of those from when the meat was cooked
fresh (data not shown). These results confirm that temperature
is a critical factor in the formation of these compounds, as
previously reported by Knize et al. (49).
The relative bioavailability of MelQx and Phi P was assessed
from the percentage of ingested amine excreted unchanged in
the urine. Samples from the first study day were collected every
4 h for 36 h to determine the time course of the urinary elim
ination of the amines. In humans, virtually all urinary excretion
of the parent amine occurs within 8 h of ingestion (limit of
detection, 4 pg/ml MelQx and 10 pg/ml PhIP; see Table 2).
Thus after the first study day, urine was collected from each
volunteer for 8 h after consumption of the standard meat meal.
To determine if the amount of free MelQx in urine changed
upon storage, samples were analyzed fresh and the results com
pared with those from duplicate samples left for 18 h at ~4°C.
The same amounts of MelQx were observed in both samples
(data not shown) and this confirms the observation of Turesky
et al. (28) that MelQx conjugates are stable in urine. When
urine samples (5 ml) in the present study were subjected to
acid hydrolysis (1 ml 0.5 M HC1, 18 h at room temperature)
the amounts of free amine determined in the samples increased
by 73 ±38% (SD) over the original values. This suggests that
some conjugates of MelQx are labile at low pH (<1.5), releasX External
6 -\
75
60
O>
C
45
0)
30
00
15
200
O)
C
'E
<0
400
600
800
1000
1200
400
TJ
0)
O)
300
u
,_
200
3
O
100
Dose
(A)
E
5 -
5000
10000
15000
Amount of amine ingested (ng)
O
Vi
3 -
Fig. 6. Linear regression analysis of MelQx (A) and PhIP (R) consumed by
each individual with the amounts of amine excreted unchanged in urine. Data are
for each subject on each study day. Correlations were highly significant for both
compounds (MelQx: r = 0.641. P < 0.001; PhIP: r = 0.686, P < 0.001).
-H
2(O
1
O)
-f-'
C
10
o
Subject
O
O)
e
O -i
5 -
Day
Fig. 5. Amount of PhIP (ti) and MelQx (•)excreted in human urine following
consumption of a cooked beef meal. Subject variation (A) and study day variation
(A). Results are mean ±SD (bars) of the percentage of estimated ingested dose.
', P< 0.01; 2-way ANOVA multiple range test using the Bonferroni method.
ing the parent amine. However, we were unable to determine
if any urinary metabolites release parent aminéas a result
of enzymatic hydrolysis, as incubation with a crude sulfatase/
glucuronidase enzyme mixture (type HI sulfatase; Sigma
Chemical Co.) led to Chromatographie interference in the assay.
A summary of the urinary excretion of unchanged MelQx
and PhIP, expressed as a percentage of the estimated dose, for
each subject is shown in Table 3. Subjects excreted an average
of 2.1% (range, 1.2-4.3%) of the MelQx and 1.1% (range,
0.6-2.3%) of the PhIP unchanged in the urine. This is similar
to the amount excreted in the rat when exposed to low doses of
MelQx or PhIP [0.01 mg/kg MelQx (40) or 0.03 mg/kg PhIP
(41), respectively]; the remainder of the absorbed dose is elim
inated by metabolism, which exhibits dose-linear kinetics.
However, at higher doses, such as those used in carcinogenicity
studies, the percentage of the dose excreted unchanged in
creases because of saturation of the pathways of metabolism.
Hence, the data in humans resemble those in the rat where
elimination is first order.
Fig. 5a shows the urinary excretion of unchanged amines as
the percentage of the dose/subject (mean ±SD). There was no
significant interday variation in MelQx or PhIP percentage
excretion (P > 0.6; 2-way ANOVA multiple range test using the
Bonferroni method), and the intraindividual CV were low for
6221
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HUMAN EXPOSURE
TO CARCINOGENS
both compounds (CV MelQx: average, 28.4%; range, 9.5-54.
1%; n = 9. CV PhIP: average, 23.7%; range, 8.7-34.4%; n = 6).
Fig. 5* shows the urinary excretion of unchanged amines as
a percentage of dose, for all volunteers, on each study day (mean
±SD). The pooled interday (intrasubject) coefficient of varia
tion was 19% for urinary excretion of MelQx and only 3.4% for
PhIP excretion (the total interval of the study was 14 months).
Thus, it should be possible to use retrospective controls in
studies involving the same subjects. Intersubject (intraday) vari
ation was greater, with a pooled coefficient of variation of 145%
for excretion of free MelQx and 71% for PhIP. Two of the
subjects excreted significantly more of the MelQx than the
others (P< 0.005, 2-way ANOVA multiple range test using the
Bonferroni method), while one of these two subjects excreted
significantly more PhIP (P< 0.01). The amounts of MelQx and
PhIP consumed by each individual were compared by linear
regression analysis with the amounts of amine excreted un
changed in urine, for each subject on each study day. There were
highly significant correlations for both compounds (MelQx:
r = 0.641 ; P < 0.001. Phi P: r = 0.686; P < 0.001 ) and results
are summarized in Fig. 6 a and b. The presence of these amines
in human urine (from healthy volunteers eating a normal diet)
has previously been demonstrated (50). The present study
shows that although there is significant interindividual variabil
ity in urinary amine excretion (expressed as a percentage of
ingested dose) the amount of amine excreted unchanged in
urine of each individual is directly proportional to the amounts
of amine consumed by that person. These studies suggest that
measurement of the percentage of excretion of MelQx and
PhIP in urine should provide an effective strategy for assessing
the relative systemic bioavailability of these amines in humans.
PhIP has recently been detected in cigarette smoke (51) and
its metabolites have been found in the urine of smokers using
•¿12P-postlabeling
following incubation of urinary extracts with
rat liver S9 mix (52). Thus systemic exposure to PhIP can arise
from several sources, including cooked meat and cigarette
smoke, both of which are associated with cancer in humans. It
is therefore vital that we determine the level of systemic expo
sure to the aminoimidazoazaarenes
and the extent to which
they are activated to their genotoxic products in vivo in humans.
It is also important that we ascertain how these are affected by
diet and other factors.
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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 Cooked Beef
Anthony M. Lynch, Mark G. Knize, Alan R. Boobis, et al.
Cancer Res 1992;52:6216-6223.
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