Environmental Health Perspectives Volume
102, Supplement 6, October 1994
[Citation
in PubMed] [Related
Articles]
Metabolism of the Food Mutagen 2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine
(PhIP) in Isolated Liver Cells from Guinea Pig, Hamster, Mouse, and Rat
Jan Alexander, Benedicte Heidenreich Fossum, and Jørn A. Holme
Department of Environmental Medicine, National Institute of Public Health,
Oslo, Norway
Abstract
The metabolism of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine
(PhIP), the most abundant compound of the aminoimidazoazaarens (AIA) group
of mutagens/carcinogens isolated from the crust of fried and broiled meat,
was examined in freshly isolated hepatocytes from untreated rat, mouse,
hamster, and guinea pig. Activation was evaluated by the total level of
covalent binding of PhIP to macromolecules. Rat hepatocytes had the lowest
rate of metabolism, both to reactive and detoxified metabolites. The products
were identified as 4´-PhIP-sulfate, PhIP-glucuronide, and N(OH)-PhIP-glucuronide.
The ring hydroxylation rate was much greater in mouse hepatocytes, the main
products being 4´-PhIP-sulfate and 4-hydroxy-PhIP. The level of covalent
binding in the mouse hepatocytes exceeded those of the rat and guinea pig
at high doses of PhIP. An extensive metabolism was seen in guinea pig hepatocytes,
the major products being 4´-PhIP-sulfate, 4´-O-PhIP glucuronide,
PhIP-glucuronide, and N(OH)-PhIP-glucuronide. In addition, several other
unknown metabolites were formed. However, the amount of covalent binding
in guinea pig hepatocytes was similar to that in rat hepatocytes. Covalent
binding of PhIP metabolites was highest in hamster hepatocytes. Three of
the main metabolites were identified as 4´-PhIP-sulfate, 4´-O-PhIP-glucuronide,
and PhIP-glucuronide, but several unknown PhIP metabolites also were formed.
Only minor amounts of N(OH)-PhIP-glucuronide were produced in the hamster.
The present study shows that both the direct detoxification of PhIP and
further conjugation of the 2-hydroxylamino-PhIP to reactive and/or detoxified
metabolites are important for the resulting covalent binding. -- Environ
Health Perspect 102(Suppl 6):109-114 (1994)
Key words: food carcinogen, PhIP, 2-amino-1-methyl-6-phenylimidazo
pyridine, hepatocytes, metabolism, species differences, rat, mouse, hamster,
guinea pig
This paper was presented at the Fifth International Conference
on Carcinogenic and Mutagenic N-Substituted Compounds held 18-21
October 1992 in Würzburg, Germany.
The technical assistance of Christine Bjørge and
Karin E. Solberg is appreciated. Benedicte H. Fossum was a fellow of the
Norwegian Council for Science and Humanities. The study was also supported
by the Norwegian Council for Agricultural Research, the latter as a part
of a Nordic collaborative project under the Scandinavian Contact Agency
for Agricultural Research.
Address correspondence to Jan Alexander, Department of
Environmental Medicine, National Institute of Public Health, Geitmyrsveien
75, N-0462 Oslo, Norway. Telephone 47 2 35 60 20. Fax 47 2 35 36 05.
Introduction
Among the carcinogenic and mutagenic aminoimidazoazaarene (AIA) group
of compounds isolated from the crust of fried meat, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine
(PhIP) is the most abundant (1-6). Although PhIP is less potent in
the Salmonella test than the quinoline- and the quinoxaline-AIA compounds,
it has been shown to be an equally potent genotoxin in mammalian cells both
in vitro and in vivo (7-10). PhIP has recently been
shown to cause colon cancer in rats (11) and abdominal lymphomas
in mice (12). In a series of previous studies, we have characterized
the metabolic pathways of PhIP leading to mutagenic activation and detoxification
(10,13-16). In these studies we have used whole rats, isolated rat
hepatocytes, subcellular fractions, and purified enzymes. Other groups have
studied PhIP activation in liver and colon tissues from humans and PhIP
metabolism in mice (17-19).
We have previously reported that 2-hydroxamino PhIP appears to be the
principal metabolite leading to mutations in Salmonella and DNA damage
in mammalian cells (10). The proximate metabolite can be further
conjugated to glucuronic acid (15). Ring hydroxylation of the phenyl
ring in position 4´ followed by sulfation appears to be the main detoxifying
pathway in the rat and the mouse (14,19). Both oxidations are P450
dependent, the rat liver P4501A2 being most active in the N-hydroxylation,
while the P4501A1 is the principal enzyme hydroxylating the ring in position
4´ (14,18).
The metabolism of PhIP will influence its genotoxic and carcinogenic
effects in vivo. Thus, characterization of the metabolism is important
for the extrapolation of effects of PhIP in animal experiments to the human
situation. We have found the use of hepatocytes in studies on metabolism
particularly valuable because this experimental system takes into account
the role of possible competing pathways and phase 2 metabolism (20,21).
In the present study, we have examined the metabolism and covalent binding
to macromolecules of PhIP in hepatocytes isolated from four different rodent
species, and we find large quantitative as well as qualitative differences
between species.
Materials and Methods
Chemicals
PhIP was donated by Dr. Errol Zeiger, National Institute of Environmental
Health (Research Triangle Park, NC). PhIP was labeled with tritium by Amersham
International plc.(Little Chalfont, UK), using their TR7 catalytic tritiation
protocol. The crude reaction mixture was purified by Lars Dragsted, Institute
of Toxicology, National Food Administration (Copenhagen, Denmark). The resulting
3H-PhIP had a specific activity of 55 mCi/mmole with a purity
of >99%. Other chemicals were obtained from the following sources: collagenase
(CLSII, 150 µ/mg) from Worthington Biochemical Corp. (Freehold, NJ);
Insta-Gel from Packard (Groningen, The Netherlands); diethylamine from BDH
Chemical Ltd (Pool, UK); bovine serum albumin (fraction V) from Sigma Chemical
Co. (St. Louis, MO). All other chemicals were commercially obtained and
of p.a. quality. Solvents for HPLC were of HPLC quality.
Animals
Male Wistar rats (MOL:WIST, 200-300 g) and male C57BL/6J Bom mice (20-30
g) were obtained from Møllegaard (Ejby, Denmark). Male Duncan-Hartley
guinea pigs (350-450 g) were obtained from J.A. Sahlin's Laboratory Animal
Farm (Malmö, Sweden). Male Syrian golden hamsters (80-100 g) were from
Wrights (Essex, Chelmsford, England). Mice, hamsters, and rats were given
Ewos R3 standard pelleted feed (Astra Ewos AB, Södertälje, Sweden)
and water ad libitum. Guinea pigs were given Ewos guinea pig pellets
and water containg 25 mg ascorbate/100 ml.
Isolation and Incubations of Hepatocytes
Liver cell suspensions were prepared by the collagenase perfusion technique
(22-24). Pooled hepatocytes from three to four mice were used in
each experiment. The hepatocytes were suspended in Hank's HEPES buffer with
1% albumin, pH 7.4, and purified by low-speed centrifugation (50g
for 30 sec). The viability was always >90%, as determined by trypan blue
exclusion. Hepatocytes (4 x 106 cells/ml) in suspensions of
2 ml were incubated for various time intervals in Hank's HEPES buffer with
1% albumin, pH 7.4.
Covalent Binding
Covalent binding of PhIP to macromolecules was determined using the glass
filter paper method (25). Aliquots of 75 µl from the hepatocyte
suspensions were transferred to Whatman GF/C glass filters (Maidstone, UK)
and macromolecules were precipitated by immersing the Þlters in 95%
ethanol, followed by washing in organic solvents. Binding was determined
using liquid scintillation counting.
HPLC Analysis
After removal of cells and precipitation of proteins with ethanol in
an argon atmosphere, the volume of the supernatant was reduced using a Speed
Vac. The metabolites were analyzed by HPLC on a Perkin-Elmer Series 4 system
using a Supelcosil DB C18 (250 x 4.6 mm i.d.) or a Waters Nova-Pak
4-µm phenyl column. The mobile phases were gradients of acetonitrile,
methanol, and water with 0.1% volume to volume ratio diethylamine adjusted
to pH 7.0 by acetic acid (details are given in the figure legends). The
eluate from the HPLC was monitored using a Hewlett-Packard 1040A photodiode
array detector. Metabolites were tentatively identified by comparison with
standards previously purified (10,13-15, unpublished data) using
retention times and UV-spectra.
Results
As previously reported (13,15), hepatocytes from untreated rats
metabolized PhIP at a relatively low rate and to few metabolites (Figure
1). Hepatocytes from mouse only produced one major metabolite, the 4´-PhIP-sulfate.
Low levels of metabolites with unknown structure were also recorded. Hamster
hepatocytes and in particular guinea pig hepatocytes transformed PhIP into
numerous metabolites. Some of these were identical to those isolated after
incubation of PhIP with hepatocytes from PCB-pretreated rats [(13,15);
unpublished data[. In all species 4´-PhIP-sulfate was the dominating
detoxication product. However, the 4´-PhIP-O-glucuronide [previously
identified in PCB-pretreated rat hepatocytes (unpublished data) and in mice
(19)] was equally abundant in the guinea pig. Three different PhIP
glucuronides yielding PhIP upon hydrolysis with ß-glucuronidase have
previously been isolated from untreated and PCB-pretreated rat hepatocytes
(unpublished data). Both rat and hamster hepatocytes produced one of these--PhIP-glucuronide3
(Figure 2). In the guinea pig hepatocytes another PhIP-glucuronide was formed,
PhIP-glucuronide2, and minor amounts of a third one, PhIP-glucuronide1 (Figure
2). PhIP-glucuronide2 was the dominating PhIP glucuronide in PCB rat hepatocytes
(unpublished data). Several unidentified metabolites of PhIP were also produced
by the hamster and guinea pig hepatocytes. Traces of two of these metabolites
were also seen in the mouse hepatocyte incubate (not shown). Unconjugated
4´-hydroxy-PhIP was seen in all incubates. The levels of unconjugated
metabolites did not seem to increase markedly with time, whereas the levels
of conjugates increased linearly with time. Larger relative amounts of 4´-hydroxy-PhIP
were seen in the hamster hepatocytes indicating saturation of conjugation
reactions in these cells (Figures 2, 3).
Figure 1. HPLC
analysis of PhIP metabolites from hepatocytes of rat (A), hamster
(B), mouse (C), and guinea pig (D). The chromatograms
show the relative metabolic profile of different species after incubating
the hepatocytes with PhIP (0.1 mM) for 2 hr. Metabolites (after removal
of cells, precipitation of proteins with ethanol, and evaporation) were
dissolved and analyzed by HPLC on a Perkin-Elmer Series 4 system using a
Waters Nova-Pak phenyl column. Metabolites were eluted by a linear gradient
from 1.5% acetonitrile and 1% methanol to 15% acetonitrile and 10% methanol
in water with 0.1% vol/vol diethylamine adjusted to pH 7.0 by acetic acid
during 60 min. Metabolite profile of guinea pig was eluted with a supralinear
gradient. Peak assignment: 1) 4´-PhIP-sulfate; 2) 4´-O-glucuronide-PhIP;
3) 4´-hydroxy-PhIP; 4) PhIP-glucuronide3; 5) N(OH)-PhIP-glucuronide;
6) PhIP-glucuronide2; 7) phenyl-substituted-PhIP; and P) PhIP. See "Note
Added in Proof."
Figure 2. Time
course of formation of PhIP metabolites of PhIP (0.1 mM) in suspensions
of hepatocytes (4 x 106 cells/ml) from rat, mouse, hamster, and
guinea pig. Values are mean of three to five experiments.
Figure 3. Concentration-dependent
formation of PhIP metabolites in suspensions of hepatocytes (4 x 106
cells/ml) incubated for 4 hr with 0.05, 0.1, and 0.5 mM PhIP. Values are
mean of three to five experiments. Hepatocytes from rat, mouse, hamster,
and guinea pig were used. Metabolite assignment as in Figure 2. See "Note
Added in Proof."
The analytical method used in these experiments did not allow determination
of the proximate metabolite 2-hydroxylamino-PhIP. However, the glucuronide
conjugate of this metabolite was a major metabolite in all species except
in the hamster, where only minor amounts could be detected (Figures 1-3).
Metabolic activation to reactive intermediates that bound covalently
to macromolecules in the hepatocytes differed greatly among the species
examined (Figure 4). At the two lower doses (0.05 and 0.1 mM) of PhIP, binding
was lowest in the rat hepatocytes, closely followed by hepatocytes from
the mouse and guinea pig, whereas a much higher binding was seen in the
hamster. At the higher dose (0.5 mM), the rat and guinea pig hepatocytes
had comparable levels of binding, whereas the binding in the mouse hepatocytes
was doubled. The hamster hepatocytes showed the highest binding of all species
studied (Figure 4).
Figure 4. Time-course
of covalent binding of PhIP (0.05, 0.1, and 0.5 mM) to macromolecules in
suspensions of hepatocytes (4 x 106 cells/ml) from rat, mouse,
hamster, and guinea pig. Values and bars are mean ± SEM of three
experiments. Bars not shown do not exceed the value mark. See "Note
Added in Proof."
Discussion
The resulting covalent binding of PhIP to macromolecules in the hepatocytes
is determined by several factors. First, activation to the proximate metabolite
2-hydroxylamino-PhIP has to take place. This is a P450-dependent reaction,
primarily involving the P4501A2, but also P4501A1, P4502C11, and a P4501A-like
enzyme (14,18,26 ). However, 2-hydroxylamino-PhIP apparently has,
unlike the 2-hydroxylamino derivatives of 2-amino-3-methylimidazo-[4,5-f
]quinoline (IQ), and 2-amino-3,8-dimethylimidazo[4,5-f ]quinoline
(MeIQx), a very low reactivity towards proteins and DNA in vitro
[(17,27,28); unpublished data]. Nevertheless, in microsomal incubations
with PhIP, protein binding occurs (19,33). One may speculate
that this may be a result of further oxidation of 2-nitro-PhIP which reacts
directly with protein sulfhydryl groups [(15); unpublished data].
Further activation of 2-hydroxylamino-PhIP, presumably by esterification
to form a leaving group, is needed in the formation of the reactive nitrenium
derivative (28,29). These pathways have not yet been characterized
for PhIP in living cells. In comparison with the quinolines IQ and MeIQ,
PhIP apparently is not dependent on the O-acetylation reaction (10).
Sulfation and other reactions may be as important (29). It is also
important to note that 2-hydroxylamino-PhIP can also be conjugated to glucuronic
acid and thus escape further activation (15). This pathway apparently
operates with low efficiency in the hamster compared with the other species
(Figures 2,3).
Metabolism by uninduced liver microsomes from rat showed nearly no detectable
oxidative activity, whereas marked N-hydroxylating activity was seen
in the guinea pig and the hamster liver microsomes, the former being more
active (not shown). Thus, lack of further activation or increased detoxification
of 2-hydroxylamino-PhIP may explain the low level of covalent binding in
guinea pig hepatocytes. In addition, the ability to detoxify directly PhIP
is also important for the resulting covalent binding and such pathways seem
to be dominant in the guinea pig in relation to its large total metabolic
activity.
Similar results were obtained in a previous study using acetylaminoþuorene
as a substrate (24). In this study it was found that with acetylaminoþuorene,
guinea pig hepatocytes had the largest ability to detoxify acetylaminofluorene
to water-soluble metabolites, and that covalent binding was highest in the
hamster hepatocytes.
In PCB-pretreated rat hepatocytes, we have previously identified a glutathione
(GSH) derivative of PhIP whose exocyclic amino group had been substituted
by GSH probably as a result of the formation of 2-nitro-PhIP (15).
Furthermore, we also identified the PhIP-cysteinylglycin derivative in the
incubations as a result of the 
-glutamyltransferase
activity on PhIP-GSH. Although GSH in general does not appear to be an important
detoxification pathway of reactive PhIP metabolites, PhIP-cysteinylglycine
was identified in the guinea pig hepatocyte incubations, indicating formation
of 2-nitro-PhIP by the guinea pig.
PhIP apparently does not induce liver cancer either in rats or in mice
(11,12), although hepatic adenoma in neonatal B6C3F1 mice (30)
have been reported. Variable results on the appearance of PhIP-induced enzyme-altered
foci in rat liver have been found (31,32). Carcinogenicity of PhIP
has not been examined in hamsters and guinea pigs. The present study on
PhIP metabolism suggests that the guinea pig would be resistant to PhIP
related to liver carcinogenesis, as it is towards acetylaminofluorene (20),
because the rate of activation relative to detoxification of PhIP is low.
In contrast, it might be expected that PhIP might cause liver cancer in
the hamster.
Note Added in Proof: Recent studies have shown that the metabolite originally
identified as a PhIP-glucuronide and designated PhIP-glucuronide2 (Figure
1, peak 6; Figures 2,3, PhIP-gluc2) is a glucuronide of 2-hydroxylamino-PhIP
different from N(OH)-PhIP-glucuronide (Figure 1, peak 5; Figures 2,3, N(OH)-PhIP-gluc).
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