Carcinogenesis vol.32 no.2 pp.233–239, 2010
Advance Access publication November 15, 2010
Rapid induction of colon carcinogenesis in CYP1A-humanized mice by
2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine and dextran sodium sulfate
Connie Cheungy, Shea Loyy, Guang Xun Li, Anna B.Liu
and Chung S.Yang?
Department of Chemical Biology, Susan Lehman Cullman Laboratory for
Cancer Research, Ernest Mario School of Pharmacy, Rutgers, The State
University of New Jersey, 164 Frelinghuysen Road, Piscataway,
NJ 08854-8020, USA
?To whom correspondence should be addressed. Tel: þ1 732 445 5360;
Fax: þ1 732 445 0687;
2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), the
most abundant heterocyclic amine produced during the cooking
of meats and fish, is suspected to be a human carcinogen. Meta-
bolic activation of PhIP is primarily mediated by the enzyme
cytochrome P450 (CYP) 1A2. Metabolism of PhIP by CYP1A2
differs considerably between humans and rodents, with more
N2-hydroxylation (activation) and less 4#-hydroxylation (detoxica-
tion) in humans. Transgenic CYP1A-humanized mice (hCYP1A-
mice), which have the human CYP1A1 and CYP1A2 genes but lack
the murine orthologs Cyp1a1 and Cyp1a2, provide an excellent
opportunity to develop a relevant model to study dietary-induced
colon carcinogenesis. The treatment with 200 mg/kg PhIP by oral
gavage, followed by 1.5% dextran sodium sulfate (DSS) in the
drinking water for7 days,was foundtobe an effective combination
to induce colon carcinogenesis in hCYP1A-mice. Tumor multiplic-
ity at week 6 was calculated to be 3.75 ± 0.70 and for week 10 was
3.90 ± 0.61 with 80–95% of the tumors being adenocarcinomas. No
tumorswere found in the similarly treated wild-type mice. Western
blots revealed overexpression of b-catenin, c-Myc, cyclin D1,
inducible nitric oxide synthaseand cyclooxygenase-2 in colon tumor
samples. Strong nuclear localization of b-catenin was observed in
tumors. These results illustrate that PhIP and DSS combination
produces rapid colon carcinogenesis in hCYP1A-mice and this is
an effective model to mimic human colon carcinogenesis.
Colorectal cancer is the third most commonly diagnosed cancer in both
men and women in the USA (1). Although the causes remain largely
unknown, dietary factors have been implicated in the etiology of this
disease, with several studies demonstrating associations between con-
sumption of fried or grilled meats with colorectal cancer (2–4).
The procarcinogen 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyr-
idine (PhIP) is the most abundant heterocyclic amine produced during
the cooking of meats and fish. The estimated daily human dietary
intake of PhIP has been reported to be 0.1–13.8 lg (5) and in another
study, 0–865 ng/day with a mean of 72 ng/day (6). Initial metabolic
activation of PhIP by N-hydroxylation is principally mediated by the
hepatic cytochrome P450 (CYP) 1A2 enzyme (7,8). CYP1A1 and
CYP1B1 can also metabolize PhIP but are considerably less active
toward PhIP than CYP1A2 (9). Following N-hydroxylation of PhIP, it
is then further conjugated to a glucuronide in the liver (10) and trans-
ported via the bile into the colon or transported as stable N-hydroxy- or
N-acetoxy-arylamines via the circulation to peripheral tissues in-
cluding the colon (11). Once reabsorbed into the colonic mucosa,
(12) or sulfotransferases (13). These activated esters can react cova-
lently with DNA and other macromolecules forming adducts that may
cause mutations and lead to the induction of cancer. A higher CYP1A2
activity in combination with higher N-acetyltransferase activity has
been associated with an elevated risk for colon cancer in individuals
eating well-cooked meats, which are a rich source of heterocyclic
PhIP induces aberrant crypt foci (ACF), which are putative pre-
cursor lesions for colon adenocarcinomas, in rats (16) and mice (17).
Studies have shown that rat colon cancers have been induced with
PhIP (18,19), whereas, in mice, administration of PhIP mainly indu-
ces non-epithelial malignancies such as malignant lymphomas and
leukemia (20–22). No reports of colon carcinogenicity from treatment
with PhIP alone exist so far (22). This suggests a weak cancer initi-
ating capability of PhIP in the colon of mice. Recent studies have
demonstrated that adenocarcinomas can be rapidly induced in the
colon of mice by combined treatment with PhIP and dextran sodium
sulfate (DSS) (23–25). DSS is a potent inducer of colitis in experi-
mental animals and DSS-induced colitis has been used as a model for
ulcerative colitis in humans (26).
Species differences in the oxidative metabolism of PhIP have
been observed between humans and rodents (27,28). In rodents,
metabolism by PhIP is predominantly oxidation in the ring system
(4#-hydroxylation) followed by Phase II conjugation. However, in
humans, N2-hydroxylation to the proximate mutagen N2-hydroxy-PhIP
is the major metabolic pathway followed by glucuronidation. Since
differences exist in the metabolism of PhIP between humans and
rodents, appropriate extrapolation of cancer risk from experimental
animals to humans is of concern, particularly in establishing safe
thresholds for human exposure to PhIP.
Humanized transgenic mice have been developed in an effort to
create more reliable in vivo systems to study and predict human
responses to xenobiotics (29). In particular, CYP1A2-humanized mice,
which express the human CYP1A2 gene but not the mouse Cyp1a2
gene, were shown to accurately express CYP1A2 protein reflective of
their expression in humans (30,31). When compared with wild-type
mice, preferential N2-hydroxylation of PhIP was demonstrated in these
CYP1A2-humanized mice, a pathway for PhIP metabolism that invitro
studies revealed as predominant with the human ortholog (30).
More recently, a transgenic mouse model, CYP1A-humanized mice
(hCYP1A-mice), was developed expressing both the human CYP1A1
and CYP1A2 genes and deficient in both the murine Cyp1a1 and
Cyp1a2 genes (32).
In studying cancer prevention and carcinogenesis, a model relevant
to human colon carcinogenesis that accurately represents the meta-
bolic activation of a dietary carcinogen such as PhIP is urgently
needed. Therefore, theprimary goal of thisstudyis todevelop amodel
forPhIP-induced coloncarcinogenesis usinghCYP1A-mice.It is anti-
cipated that the increased metabolic activation of PhIP in hCYP1A-
mice (30) will result in greater carcinogenic effect when compared
with wild-type mice. Colon cancer is expected to be the predominant
cancer in mice given a combination of PhIP and DSS as observed in
previous studies (23–25), whereas, other types of cancer such as pros-
tate cancer and mammary cancer may also develop in micegivenPhIP
alone. The hCYP1A-mice may also be a good model for other can-
cers. The results described herein, demonstrate that a PhIP and DSS
treatment combination to hCYP1A-mice provides a more effective
model of colon carcinogenesis when compared with wild-type mice.
Colon tumors are shown to be optimally formed at 6–10 weeks after
a treatment with 200 mg/kg PhIP followed by 1.5% DSS. The PhIP-
induced colon carcinogenesis model using hCYP1A-mice presents an
excellent model for colon cancer prevention studies.
Abbreviations: ACF, aberrant crypt foci; COX-2, cyclooxygenase-2; CYP,
cytochrome P450; DMSO, dimethyl sulfoxide; DSS, dextran sodium sulfate;
iNOS, inducible nitric oxide synthase; PhIP, 2-amino-1-methyl-6-phenylimi-
yThese authors contributed equally to this work.
Published by Oxford University Press 2010.
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Received June 22, 2010; revised November 2, 2010;
accepted November 7, 2010
Colon carcinogenesis by PhIP in hCYP1A-mice