0090-9556/06/3410-1697–1702$20.00
DRUG METABOLISM AND DISPOSITION
Copyright © 2006 by The American Society for Pharmacology and Experimental Therapeutics
DMD 34:1697–1702, 2006
Vol. 34, No. 10
10819/3137895
Printed in U.S.A.
In Vivo and in Vitro Metabolism of Arylamine Procarcinogens in
Acetyltransferase-Deficient Mice
K. S. Sugamori, D. Brenneman, and D. M. Grant
Department of Pharmacology, Faculty of Medicine (K.S.S., D.B., D.M.G.), and Leslie Dan Faculty of Pharmacy (D.M.G.),
University of Toronto, Toronto, Ontario, Canada
Received April 25, 2006; accepted June 27, 2006
ABSTRACT:
phenyl (ABP) and 2-aminofluorene (AF), whereas significant levels
were measured in all wild-type tissue cytosols tested, indicating
the widespread metabolism of these agents. Nat1/2(ⴚ/ⴚ) mice
displayed a variable response with respect to in vivo pharmacokinetics. AF appeared to be most severely compromised, with a 3- to
4-fold increased area under the curve (AUC), whereas the clearance of ABP was found to be less dependent on N-acetylation, with
no difference in ABP-AUC between wild-type and knockout animals. 2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine was neither N-acetylated nor was its clearance affected by NAT genotype,
signifying a dependence on other drug-metabolizing enzymes. The
elucidation of the role that N-acetylation plays in the clearance of
procarcinogenic agents is the first step in attempting to correlate
metabolism by NATs to toxic outcome prevention or augmentation.
Occupational or environmental exposure to arylamines has long
been considered a causal factor for the development of human cancers
(Weisburger, 2002). A necessary prerequisite for these chemicals to
exert their deleterious effects is biotransformation by various drugmetabolizing enzymes. In particular, the polymorphic arylamine Nacetyltransferases (NATs) catalyze the acetylation of these and other
aromatic and heterocyclic amine drugs. Two isoenzymes, NAT1 and
NAT2, are present in humans and exhibit unique tissue expression
profiles and somewhat distinct but overlapping substrate specificities
(for review, see Grant et al., 2000; Butcher et al., 2002). Paradoxically, NATs can either facilitate the detoxification of carcinogenic
arylamines into innocuous metabolites by N-acetylation or promote
their metabolic activation into DNA-binding electrophiles via Oacetylation (Hein, 2002). Such procarcinogenic arylamines include
the bladder cancer agent 4-aminobiphenyl (ABP), which is found in
tobacco smoke (Stabbert et al., 2003) and commercial hair dyes
(Turesky et al., 2003), and 2-aminofluorene (AF), an agent whose
N-acetylated derivative was originally developed as an insecticide
(Heflich and Neft, 1994), but which is used now only as a model
compound for studying mechanisms of metabolic activation in chemically induced mutagenesis and carcinogenesis. In addition, heterocyclic aromatic amines found in grilled meats, poultry, and fish, such as
2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), are considered as potential etiologic agents in human cancers (Weisburger,
2002).
The following two-step activation model has been proposed for the
conversion of procarcinogenic arylamines into DNA-binding molecules. The arylamine is first N-oxidized to an arylhydroxylamine by
one or more isoforms of cytochrome P450, such as CYP1A2 present
in the liver (see Kim and Guengerich, 2005). Subsequent O-conjugation by NATs leads to the formation of highly unstable acetoxy esters
that decompose into DNA-binding arylnitrenium ions (Hein, 2002).
Competing with the N-oxidation in the liver is N-acetylation by NATs,
a step that converts potentially carcinogenic arylamines into N-acetate
metabolites which are, in many cases, less carcinogenic than the
parent compound. The balance of these pathways and their cellspecific expression profiles may thus determine the toxic endpoint in
a particular tissue upon arylamine exposure.
Numerous studies have documented either the extent of DNA
binding, mutational frequency and spectra, or the tumorigenic potential of ABP, AF, or PhIP in various animal models (Schieferstein et
al., 1985; Flammang et al., 1992; Levy and Weber, 1992; Poirier et al.,
1995; Fletcher et al., 1998), human tissue samples (Airoldi et al.,
This research was supported by an operating grant from the National Cancer
Institute of Canada, with funding from the Canadian Cancer Society.
Article, publication date, and citation information can be found at
http://dmd.aspetjournals.org.
doi:10.1124/dmd.106.010819.
ABBREVIATIONS: ABP, 4-aminobiphenyl; AABP, N-acetyl-4-aminobiphenyl; AF, 2-aminofluorene; AAF, N-acetyl-2-aminofluorene; NAT, Nacetyltransferase; PhIP, 2-amino-1-methyl-6-phenyl[4,5-b]pyridine; APhIP, N-acetyl-PhIP; HPLC, high-performance liquid chromatography; AUC,
area under the curve.
1697
Downloaded from dmd.aspetjournals.org at ASPET Journals on May 9, 2016
Arylamine N-acetyltransferases (NATs) catalyze the biotransformation of a number of aromatic and heterocyclic amines, many of
which are procarcinogenic agents. Interestingly, these enzymes
are binary in nature, participating in both detoxification and activation reactions, and thus it is unclear what role NATs actually play
in either preventing or enhancing toxic responses. The ultimate
direction may be substrate-specific and dependent on its tissuespecific metabolism by competing, but genetically variable, drugmetabolizing enzymes. To investigate the effect of N-acetylation
on the metabolism of some classical procarcinogenic arylamines,
we have used our double knockout Nat1/2(ⴚ/ⴚ) mouse model to
test both in vitro activity and the in vivo clearance of some of these
agents. As expected, N-acetylation activity was undetectable in
tissue cytosol preparations from Nat1/2(ⴚ/ⴚ) mice for 4-aminobi-
1698
SUGAMORI ET AL.
system composed of 5 mM acetyl-DL-carnitine and 1 U of carnitine
acetyltransferase per milliliter of assay buffer (250 mM triethanolamine-HCl, 5 mM EDTA, 5 mM dithiothreitol, pH 7.5), and 50 ␮l of
diluted cytosol at a protein concentration of 1 ␮g/␮l. Reactions were
initiated with the addition of the cytosol to the remaining preheated
components, incubated for 10 min at 37°C, and terminated by the
addition of 10 ␮l of 15% perchloric acid. For PhIP, the reactions were
initiated with either 50 or 100 ␮g of cytosolic protein and were
allowed to proceed for 60 min at 37°C with up to 0.5 mM PhIP. After
precipitation of the denatured protein, the supernatant fractions were
assayed for the N-acetylated product (AABP, AAF, or APhIP) by
HPLC using a Shimadzu LC-2010A system (Shimadzu Scientific
Instruments Inc., Columbia, MD) and a reverse-phase Beckman Ultrasphere ODS 5-␮m column (15 cm ⫻ 4.6 mm i.d.; Beckman
Coulter, Fullerton, CA). A flow rate of 2 ml/min with a mobile phase
consisting of 66% sodium perchlorate buffer (20 mM, pH 2.5)/34%
acetonitrile and an ultraviolet detector setting of 280 nm were used for
ABP and AF. For PhIP, the mobile phase consisted of 85% sodium
perchlorate buffer/15% acetonitrile and the ultraviolet detector setting
was 317 nm. Under these conditions, retention times of parent compound and acetylated metabolite were, respectively, 1.4 min and 3.7
min (AF and AAF), 1.4 min and 3.3 min (ABP and AABP), and 6.8
min and 6.2 min (PhIP and APhIP).
Drug Administration and Plasma Elimination Kinetics. ABP
(50 mg/kg), AF (50 mg/kg), or PhIP (50 mg/kg) dissolved in dimethyl
sulfoxide was administered by i.p. injection to 8- to 9-week-old ageand sex-matched Nat1/2(⫺/⫺) mice (n ⫽ 4) and wild-type sibs (n ⫽
4). Blood samples were drawn from the saphenous vein using hepa-
Materials and Methods
Materials. F2 Nat1/2(⫹/⫹) and Nat1/2(⫺/⫺) mice on a mixed
genetic B6/129 background were generated as described previously
(Sugamori et al., 2003). All procedures involving animals were used
in accordance with the Canadian Council for Animal Care guidelines.
Acetyl-DL-carnitine, carnitine acetyltransferase, acetyl-CoA sodium
salt, ABP, AF, and AAF used for NAT activity assays were acquired
from Sigma-Aldrich Canada Ltd. (Oakville, ON, Canada). AABP to
use as a standard was produced in definable quantities from ABP
using wild-type recombinant human NAT1 as a catalyst (Sugamori et
al., 2003). PhIP was purchased from Toronto Research Chemicals
(Toronto, ON, Canada), and APhIP was generously provided by Dr.
M. Novak (Miami University, Oxford, OH).
In Vitro NAT Enzyme Activity. Tissue cytosols were prepared
from Nat1/2(⫹/⫹) and Nat1/2(⫺/⫺) animals as described (Sugamori
et al., 2003). NAT activity for ABP and AF N-acetylation was
determined using various tissue cytosols (n ⫽ 3 per gender, genotype,
and tissue). Initial enzyme velocities for cytosolic fractions were
performed in duplicate in final reaction volumes of 100 ␮l, each
consisting of 10 ␮l of ABP or AF solution (giving a final concentration of 0.1 mM), 20 ␮l of 0.5 mM acetyl-CoA (giving a final
concentration of 0.1 mM), 20 ␮l of an acetyl-CoA regenerating
FIG. 1. N-Acetylation rates in cytosols from wild-type and Nat1/2(⫺/⫺) mice.
Tissue cytosols from wild-type and Nat1/2(⫺/⫺) mice were assayed for NAT
activity with either 0.1 mM AF (A) or 0.1 mM ABP (B) in the presence of 0.1 mM
acetyl-CoA as described under Materials and Methods. AAF and AABP were
separated and quantified by HPLC. Product formation rates represent the mean from
n ⫽ 3 wild-type or knockout animals, respectively. Cortex ⫽ cerebral cortex of the
brain. Detection limits for N-acetylated product formation rates were 0.01, 0.02, and
0.02 nmol/min/mg for AAF, AABP, and APhIP, respectively.
Downloaded from dmd.aspetjournals.org at ASPET Journals on May 9, 2016
2002; Faraglia et al., 2003), cell cultures (Feng et al., 2002), and
NAT-expressing Salmonella typhimurium strains (Grant et al., 1992;
Oda, 2004). Results from these studies clearly indicate that these
agents are mutagenic and/or require metabolic activation. Furthermore, epidemiological studies have alluded to a link between acetylation status and certain cancers, particularly of the colon and bladder
(see Hein et al., 2000, for review), but results have been inconsistent
because of differences in sample size, genotyping/phenotyping discordances, and variable exposure situations.
Whereas epidemiological and in vitro studies have proposed a link
between various drug-metabolizing enzymes as either risk modifiers
or mediators of increased mutagenicity, results from whole animal
studies involving knockout mouse models have revealed some unexpected results. In particular, Cyp1a2 knockout mice do not have lower
ABP-DNA adducts (Tsuneoka et al., 2003) or attenuated ABP-induced methemoglobinemia (Shertzer et al., 2002), nor do these animals display an increased protection against either ABP (Kimura et
al., 1999)- or PhIP-induced carcinogenesis (Kimura et al., 2003).
Furthermore, Cyp1a1 knockout mice exhibited a slower rate of clearance of benzo[a]pyrene upon oral exposure but a much greater toxicity (Uno et al., 2004), despite in vitro studies that indicate a facilitatory role for CYP1A1 in benzo[a]pyrene toxicity (Schmalix et al.,
1993). These studies undoubtedly emphasize the importance of performing in vivo studies in conjunction with in vitro studies to be able
to fully understand the potential interplay between multiple enzyme
systems, their tissue-specific expression patterns, and the effect of
clearance on activation.
Using a mouse model deficient in both NAT1 and NAT2, namely,
our Nat1/2(⫺/⫺) model (Sugamori et al., 2003), we have investigated
the effect of acetylation on the metabolism and clearance of the
procarcinogens ABP, AF, and PhIP. This is a necessary step in
dissecting out the importance of acetylation on the metabolism and
clearance of these compounds in the whole animal. Although NAT
knockout models (Cornish et al., 2003; Sugamori et al., 2003) do not
exhibit a deleterious phenotype, which would be indicative of a
potential developmental role, these models are extremely useful in
toxicity/carcinogenicity testing (Gonzalez, 2003), and for delineating
the relative contribution of various enzyme biotransformation reactions to detoxification and/or bioactivation in the whole animal.
NAT FUNCTION IN GENETICALLY MODIFIED MICE
1699
rinized Microvettes (Sartstedt Inc., Montreal, QC, Canada) at four
sequential time points and centrifuged to separate plasma. The time
points for AF plasma sampling were 2, 6, 22, and 24 h for female
knockout mice, and 1, 2, 4, and 6 h for female wild-type, male
wild-type, and male knockout mice. ABP plasma sampling times were
2, 6, 22, and 24 h for both genders and all genotypes. PhIP plasma
sampling times were 30 min, 60 min, 90 min, and 120 min for all
animals. Plasma samples were diluted 1:50 in HPLC mobile phase,
and analyzed for parent and acetylated metabolites by HPLC as
described above. Blank plasma samples were spiked with known
amounts of AF and AAF, ABP and AABP, or PhIP and APhIP to
quantify the amount of parent and acetylated products present in the
samples. Area under the curve (AUC) values were determined by the
trapezoidal rule using the computer program Prism (GraphPad Software Inc., San Diego, CA). Statistical analyses were performed using
a Student’s t test with GraphPad Prism.
Results
ABP and AF in Vitro NAT Enzyme Activity. Nat1/2(⫺/⫺) mice
displayed no measurable cytosolic ABP- or AF-NAT activity in any
tissue assayed, whereas their wild-type controls displayed quantifiable
NAT activity in all tissues (Fig. 1). A gender difference in acetylation
using kidney cytosol was noted in the B6/129 F2 wild-type mice.
Male kidney cytosol displayed about a 2-fold higher activity ( p ⬍
0.001) than female kidney cytosol for both AF and ABP. No other
tissue cytosols displayed a gender difference in NAT activity in the
wild-type animals. No PhIP N-acetylation could be detected in liver
tissue cytosol from either wild-type or knockout animals (data not
shown).
In Vivo NAT Activity and Plasma Pharmacokinetics. Consistent
with the in vitro results, Nat1/2(⫺/⫺) mice exhibited no ability to
N-acetylate either ABP or AF upon in vivo administration. Clearance
for ABP appeared to be less dependent on the formation of its
N-acetylated metabolite, as determined by the small AUC values for
AABP compared with ABP in the wild-type animals (Fig. 2, A and C)
and the lack of a significant change in the ABP-AUC values in the
knockout animals compared with wild-type (Table 1). Knockout animals for both genders did display a complete absence of AABP (Fig.
2, B and D). The lack of a significant difference between wild-type
and knockout animals for ABP-AUC values indicates that N-acetylation does not appear to play a major role in the in vivo elimination of
ABP. In addition, AABP was not detected in urine samples from
wild-type animals (data not shown).
In contrast, the absence of functional NAT1 and NAT2 appeared to
impair the clearance of AF, with ⬃3- to 4-fold higher AF-AUC values
from the knockout animals compared with wild-type for both genders
(Figs. 3 and 4), although the mean AF-AUC value for wild-type
females was approximately 3-fold higher than that for wild-type males
(Table 1, p ⬍ 0.05). Knockout animals displayed a complete absence
of N-acetylated metabolite in all plasma samples analyzed (Fig. 3, B
and D). The small AUC values for AAF compared with AF may
indicate that this metabolite is rapidly cleared from plasma.
N-acetylated PhIP could not be detected in the plasma samples from
either the wild-type or knockout animals (Fig. 5). AUC values for the
parent PhIP were not significantly different between wild-type and
knockout animals (Table 1). The smaller AUC values for PhIP compared with either AF or ABP, however, indicate that PhIP is rapidly
eliminated from plasma, presumably by other pathways that may
include redistribution into other tissue compartments or efficient renal
clearance.
Downloaded from dmd.aspetjournals.org at ASPET Journals on May 9, 2016
FIG. 2. Plasma pharmacokinetics of ABP
and AABP in wild-type and Nat1/2(⫺/⫺)
mice. A, plasma kinetics of ABP and
AABP from a representative wild-type
male mouse. AUC values for ABP and
AABP were 286.5 and 7.0 mmol min/l. B,
plasma kinetics of ABP and AABP for a
representative knockout male mouse. AUC
for ABP was 266.1 mmol min/l. C, plasma
kinetics of ABP and AABP from a representative wild-type female mouse. AUC
values for ABP and AABP were 205.4 and
6.6 mmol min/l, respectively. D, plasma
kinetics of ABP and AABP from a representative knockout female mouse. AUC
value for ABP was 223.7 mmol min/l. For
all mice, ABP (50 mg/kg) was administered
by i.p. injection, and four sequential blood
samples were drawn from the saphenous
vein of each mouse at the indicated time
points. Plasma was isolated from each
blood sample, diluted 1:50, and analyzed
for ABP and AABP by HPLC. AUC values
were determined by the trapezoidal rule
using the computer program Prism (GraphPad Software Inc.). Detection limit for the
N-acetylated metabolite was 4 ␮M.
1700
SUGAMORI ET AL.
TABLE 1
AUC values for ABP, AF, and PhIP elimination
AUC values (mmol min l⫺1, mean ⫾ S.D., n ⫽ 4) were determine by the trapezoidal rule using Prism software (GraphPad Software Inc.). ABP, AF, or PhIP (50 mg/kg) was dosed by i.p.
injection to age- and sex-matched wild-type and knockout mice. Four sequential blood samples were drawn and analyzed for parent and metabolite compounds as described under Materials
and Methods.
Genotype
ABP
AcABP
AF
AcAF
PhIP
APhIP
Male ⫹/⫹
Male ⫺/⫺
Female ⫹/⫹
Female ⫺/⫺
219 ⫾ 72
264 ⫾ 64
195 ⫾ 8
190 ⫾ 30
5.8 ⫾ 0.9**
0
7.1 ⫾ 1.9**
0
24.7 ⫾ 4.8*
85.8 ⫾ 30.0
71.7 ⫾ 36.2*
314 ⫾ 124
2.3 ⫾ 0.9*
0
4.0 ⫾ 2.1*
0
5.4 ⫾ 0.7
5.2 ⫾ 1.3
6.3 ⫾ 0.8
7.3 ⫾ 1.2
0
0
0
0
* Significantly different from knockout mice of the same gender ( p ⬍ 0.05).
** Significantly different from knockout mice of the same gender ( p ⬍ 0.001).
Discussion
The double Nat1/2 knockout model was used to investigate the
effect of N-acetylation on the metabolism of the aromatic and heterocyclic amine procarcinogens, ABP, AF, and PhIP. In vitro enzyme
assays using various tissue cytosols from the Nat1/2(⫺/⫺) revealed a
complete absence of either N-acetylated ABP or AF. In contrast, the
N-acetylated metabolites of ABP or AF were observed in all tissues
assayed from wild-type animals, indicating the widespread functional
expression of NAT. Most notably, NAT activity was observed in the
bladder, a site susceptible to the formation of tumors upon ABP
administration. No N-acetylation of PhIP could be detected even in
our wild-type animals, indicating that PhIP is not a good substrate for
N-acetylation by the mouse enzymes.
Consistent with our previous results using p-aminosalicylate as a
substrate (Sugamori et al., 2003) and those using p-aminobenzoic acid
and AF (Glowinski and Weber, 1982; Smolen et al., 1993), a gender
difference was also observed in our wild-type mice with respect to
kidney ABP- and AF-NAT activity. The activity in male kidney has
been shown to be modulated by testosterone (Smolen et al., 1993) and
is not unique to NATs inasmuch as CYP2E1 activity has also been
shown to be higher in male kidney (Hoivik et al., 1995). Whether this
higher activity in kidney translates to any gender-related differences
in arylamine adduct levels and a differential susceptibility to
arylamine-induced carcinogenesis warrants further investigation. Indeed, gender differences have been observed with respect to the
formation of ABP- and AF-DNA adducts in the bladder and the
incidence of ABP-induced bladder cancers. In general, male mice
were found to have higher DNA adducts in the bladder and a higher
bladder tumor incidence (Schieferstein et al., 1985; Flammang et al.,
1992; Levy and Weber, 1992; Poirier et al., 1995; Tsuneoka et al.,
2003). Female mice, on the other hand, had higher liver DNA adduct
levels (Levy and Weber, 1989; Poirier et al., 1995; Tsuneoka et al.,
2003; Chen et al., 2005) and a higher incidence of liver cancer
(Schieferstein et al., 1985; Poirier et al., 1995).
Downloaded from dmd.aspetjournals.org at ASPET Journals on May 9, 2016
FIG. 3. Plasma pharmacokinetics of AF
and AAF in wild-type and Nat1/2(⫺/⫺)
mice. A, plasma kinetics of AF and AAF
from a representative wild-type male
mouse. AUC values for AF and AAF were
28.5 and 2.0 mmol min/l. B, plasma kinetics of AF and AAF from a representative
knockout male mouse. AUC for AF was
72.9 mmol min/l. C, plasma kinetics of AF
and AAF from a representative wild-type
female mouse. AUC values for AF and
AAF were 54.7 and 5.5 mmol min/l, respectively. D, plasma kinetics of AF and
AAF from a representative knockout female mouse. AUC value for AF was 251
mmol min/l. For all mice, AF (50 mg/kg)
was administered by i.p. injection, and four
sequential blood samples were drawn from
the saphenous vein of each mouse at the
indicated time points. Plasma was isolated
from each blood sample, diluted 1:50 and
analyzed for AF and AAF by HPLC. AUC
values were determined by the trapezoidal
rule using the computer program Prism
(GraphPad Software Inc.). Detection limit
for the N-acetylated metabolite was 4 ␮M.
NAT FUNCTION IN GENETICALLY MODIFIED MICE
FIG. 4. Graphical representation of AUC values for knockout animals plotted as a
percentage of wild-type AUC. Values for p-aminosalicylate (PAS) and sulfamethazine (SMZ) were taken from Sugamori et al. (2003).
The clearance of PhIP was not dependent on N-acetylation. AUC
values for PhIP were not significantly different between wild-type and
knockout animals. Acetylated PhIP could not be detected in any of the
plasma samples, confirming previous results in which PhIP was found
to be a poor substrate for N-acetylation (Minchin et al., 1992).
However, the rather small AUC for the parent compound compared
with AF and ABP suggests that PhIP is rapidly cleared from plasma.
This would implicate the involvement either of other drug-metabolizing enzymes or enzymatic reactions in the clearance of PhIP from
mice, redistribution into another poorly accessible tissue compartment, or efficient renal elimination. PhIP has been shown to be
preferentially metabolized by 4⬘-hydroxylation in wild-type mice, a
step that is considered to be a detoxification step with 4⬘-PhIP-sulfate,
a main urinary product formed (Buonarati et al., 1992), whereas in
humanized CYP1A2 mice, N2-hydroxylation or activation was found
to be the predominant pathway (Cheung et al., 2005). This may
account for the generally higher tumor incidence in Cyp1a2(⫺/⫺)
mice exposed to PhIP (Kimura et al., 2003), since 4⬘-hydroxylation or
detoxification is a major pathway in wild-type mice, and thus,
CYP1A2 may actually have a more protective role in wild-type mice.
The results using our double Nat1/2 knockout model indicate that
the procarcinogens ABP, AF, and PhIP display a differential dependence on N-acetylation for clearance. What effect this may have with
respect to the formation of DNA adducts and tumor incidence remains
to be determined. Studies using congenic rapid and slow Nat2 acetylator mice and a transgenic mouse model expressing human NAT1
found no correlation between murine NAT2 activity or human NAT1
expression and ABP-DNA adduct levels in liver (McQueen et al.,
2003). It was proposed that the activation of ABP may be masked by
competing reactions since ABP is a substrate for both NAT1 and
NAT2 (Fretland et al., 1997), and thus, a mouse model lacking both
enzymes, such as our Nat1/2(⫺/⫺) mice, may be needed to assess the
full impact of variation of acetylation capacity to the formation of
ABP-DNA adducts. However, if clearance does affect the extent of
FIG. 5. Plasma pharmacokinetics of PhIP in wild-type and
Nat1/2(⫺/⫺) mice. A, plasma kinetics of PhIP and APhIP
from a representative wild-type male mouse. AUC value for
PhIP was 5.0 mmol min/l. B, plasma kinetics of PhIP and
APhIP from a representative knockout male mouse. AUC
for PhIP was 4.9 mmol min/l. C, plasma kinetics of PhIP and
APhIP from a representative wild-type female mouse. AUC
value for PhIP was 6.3 mmol min/l, respectively. D, plasma
kinetics of PhIP and APhIP from a representative knockout
female mouse. AUC value for PhIP was 6.3 mmol min/l. For
all mice, PhIP (50 mg/kg) was administered by i.p. injection,
and four sequential blood samples were drawn from the
saphenous vein of each mouse at the indicated time points.
Plasma was isolated from each blood sample, diluted 1:50,
and analyzed for PhIP and APhIP by HPLC. AUC values
were determined by the trapezoidal rule using the computer
program Prism (GraphPad Software Inc.). Detection limit
for the N-acetylated metabolite was 10 ␮M.
Downloaded from dmd.aspetjournals.org at ASPET Journals on May 9, 2016
To determine the effect of the absence of functional NAT1 and
NAT2 activity on the clearance of ABP, AF, and PhIP, in vivo
elimination studies were undertaken. The clearance of ABP from
plasma was essentially unchanged in the Nat1/2(⫺/⫺) mice, thereby
indicating that N-acetylation does not appear to be a major determinant of the clearance of ABP in the mouse. Contrary to ABP, the in
vivo clearance of AF was found to be impaired in the Nat1/2(⫺/⫺)
mice, as determined by the 3- to 4-fold increase in AF-AUC values for
the knockout animals compared with wild-type. Although very low
levels of AF N-acetylation have been observed with recombinant
NAT3 (Kelly and Sim, 1994), no detectable AAF was observed in our
knockout animals using both in vitro activity assays and in vivo
elimination studies. Clearance for AF appeared to be slower in female
mice, regardless of genotype. It remains to be seen whether the
previously observed gender-related differences in liver AF-DNA adducts, namely, that female mice have higher hepatic AF-DNA adducts, is related to this difference in clearance.
1701
1702
SUGAMORI ET AL.
Acknowledgments. We thank Roopam Singh and Simin Yazdanpanah for excellent technical assistance.
References
Airoldi L, Orsi F, Magagnotti C, Coda R, Randone D, Casetta G, Peluso M, Hautefeuille A,
Malaveille C, and Vineis P (2002) Determinants of 4-aminobiphenyl-DNA adducts in bladder
cancer biopsies. Carcinogenesis 23:861– 866.
Buonarati MH, Roper M, Morris CJ, Happe JA, Knize MG, and Felton JS (1992) Metabolism of
2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) in mice. Carcinogenesis 13:621–
627.
Butcher NJ, Boukouvala S, Sim E, and Minchin RF (2002) Pharmacogenetics of the arylamine
N-acetyltransferases. Pharmacogenomics J 2:30 – 42.
Chen T, Mittelstaedt RA, Beland FA, Heflich RH, Moore MM, and Parsons BL (2005)
4-Aminobiphenyl induces liver DNA adducts in both neonatal and adult mice but induces liver
mutations only in neonatal mice. Int J Cancer 117:182–187.
Cheung C, Ma X, Krausz KW, Kimura S, Feigenbaum L, Dalton TP, Nebert DW, Idle JR, and
Gonzalez FJ (2005) Differential metabolism of 2-amino-1-methyl-6-phenylimidazo[4,5b]pyridine (PhIP) in mice humanized for CYP1A1 and CYP1A2. Chem Res Toxicol 18:1471–
1478.
Cornish VA, Pinter K, Boukouvala S, Johnson N, Labrousse C, Payton M, Priddle H, Smith AJ,
and Sim E (2003) Generation and analysis of mice with a targeted disruption of the arylamine
N-acetyltransferase type 2 gene. Pharmacogenomics J 3:169 –177.
Faraglia B, Chen SY, Gammon MD, Zhang Y, Teitelbaum SL, Neugut AI, Ahsan H, Garbowski
GC, Hibshoosh H, Lin D, et al. (2003) Evaluation of 4-aminobiphenyl-DNA adducts in human
breast cancer: the influence of tobacco smoke. Carcinogenesis 24:719 –725.
Feng Z, Hu W, Rom WN, Beland FA, and Tang MS (2002) 4-Aminobiphenyl is a major
etiological agent of human bladder cancer: evidence from its DNA binding spectrum in human
p53 gene. Carcinogenesis 23:1721–1727.
Flammang TJ, Couch LH, Levy GN, Weber WW, and Wise CK (1992) DNA adduct levels in
congenic rapid and slow acetylator mouse strains following chronic administration of 4-aminobiphenyl. Carcinogenesis 13:1887–1891.
Fletcher K, Tinwell H, and Ashby J (1998) Mutagenicity of the human bladder carcinogen
4-aminobiphenyl to the bladder of MutaMouse transgenic mice. Mutat Res 400(1–2):245–250.
Fretland AJ, Devanaboyina US, Doll MA, Zhao S, Xiao GH, and Hein DW (2003) Metabolic
activation of 2-hydroxyamino-1-methyl-6-phenylimidazo[4,5-b]pyridine in Syrian hamsters
congenic at the N-acetyltransferase 2 (NAT2) locus. Toxicol Sci 74:253–259.
Fretland AJ, Doll MA, Gray K, Feng Y, and Hein DW (1997) Cloning, sequencing, and
recombinant expression of NAT1, NAT2, and NAT3 derived from the C3H/HeJ (rapid) and
A/HeJ (slow) acetylator inbred mouse: functional characterization of the activation and
deactivation of aromatic amine carcinogens. Toxicol Appl Pharmacol 142:360 –366.
Glowinski IB and Weber WW (1982) Biochemical characterization of genetically variant
aromatic amine N-acetyltransferases in A/J and C57BL/6J mice. J Biol Chem 257:1431–1437.
Gonzalez FJ (2003) Role of gene knockout and transgenic mice in the study of xenobiotic
metabolism. Drug Metab Rev 35:319 –335.
Grant DM, Goodfellow GH, Sugamori K, and Durette K (2000) Pharmacogenetics of the human
arylamine N-acetyltransferases. Pharmacology 61:204 –211.
Grant DM, Josephy PD, Lord HL, and Morrison LD (1992) Salmonella typhimurium strains
expressing human arylamine N-acetyltransferases: metabolism and mutagenic activation of
aromatic amines. Cancer Res 52:3961–3964.
Heflich RH and Neft RE (1994) Genetic toxicity of 2-acetylaminofluorene, 2-aminofluorene and
some of their metabolites and model metabolites. Mutat Res 318:73–114.
Hein D (2002) Molecular genetics and function of NAT1 and NAT2: role in aromatic amine
metabolism and carcinogenesis. Mutat Res 506 –507(C):65–77.
Hein DW, Doll MA, Fretland AJ, Leff MA, Webb SJ, Xiao GH, Devanaboyina US, Nangju NA,
and Feng Y (2000) Molecular genetics and epidemiology of the NAT1 and NAT2 acetylation
polymorphisms. Cancer Epidemiol Biomarkers Prev 9:29 – 42.
Hoivik DJ, Manautou JE, Tveit A, Hart SG, Khairallah EA, and Cohen SD (1995) Gender-related
differences in susceptibility to acetaminophen-induced protein arylation and nephrotoxicity in
the CD-1 mouse. Toxicol Appl Pharmacol 130:257–271.
Kelly SL and Sim E (1994) Arylamine N-acetyltransferase in BALB/c mice: identification of a
novel mouse isoenzyme by cloning and expression in vitro. Biochem J 302(Pt 2):347–353.
Kim D and Guengerich FP (2005) Cytochrome P450 activation of arylamines and heterocyclic
amines. Annu Rev Pharmacol Toxicol 45:27– 49.
Kimura S, Kawabe M, Ward JM, Morishima H, Kadlubar FF, Hammons GJ, Fernandez-Salguero
P, and Gonzalez FJ (1999) CYP1A2 is not the primary enzyme responsible for 4-aminobiphenyl-induced hepatocarcinogenesis in mice. Carcinogenesis 20:1825–1830.
Kimura S, Kawabe M, Yu A, Morishima H, Fernandez-Salguero P, Hammons GJ, Ward JM,
Kadlubar FF, and Gonzalez FJ (2003) Carcinogenesis of the food mutagen PhIP in mice is
independent of CYP1A2. Carcinogenesis 24:583–587.
Lai CC, Miller EC, Miller JA, and Liem A (1987) Initiation of hepatocarcinogenesis in infant
male B6C3F1 mice by N-hydroxy-2-aminofluorene or N-hydroxy-2-acetylaminofluorene depends primarily on metabolism to N-sulfooxy-2-aminofluorene and formation of DNA(deoxyguanosin-8-yl)-2-aminofluorene adducts. Carcinogenesis 8:471– 478.
Levy GN and Weber WW (1989) 2-Aminofluorene-DNA adduct formation in acetylator congenic mouse lines. Carcinogenesis 10:705–709.
Levy GN and Weber WW (1992) 2-Aminofluorene-DNA adducts in mouse urinary bladder:
effect of age, sex and acetylator phenotype. Carcinogenesis 13:159 –164.
McQueen CA, Chau B, Erickson RP, Tjalkens RB, and Philbert MA (2003) The effects of
genetic variation in N-acetyltransferases on 4-aminobiphenyl genotoxicity in mouse liver.
Chem Biol Interact 146:51– 60.
Miller JA (1994) Sulfonation in chemical carcinogenesis— history and present status. Chem Biol
Interact 92(1–3):329 –341.
Minchin RF, Reeves PT, Teitel CH, McManus ME, Mojarrabi B, Ilett KF, and Kadlubar FF
(1992) N-and O-acetylation of aromatic and heterocyclic amine carcinogens by human
monomorphic and polymorphic acetyltransferases expressed in COS-1 cells. Biochem Biophys
Res Commun 185:839 – 8442.
Oda Y (2004) Analysis of the involvement of human N-acetyltransferase 1 in the genotoxic
activation of bladder carcinogenic arylamines using a SOS/umu assay system. Mutat Res
554(1–2):399 – 406.
Poirier MC, Fullerton NF, Smith BA, and Beland FA (1995) DNA adduct formation and
tumorigenesis in mice during the chronic administration of 4-aminobiphenyl at multiple dose
levels. Carcinogenesis 16:2917–2921.
Schieferstein GJ, Littlefield NA, Gaylor DW, Sheldon WG, and Burger GT (1985) Carcinogenesis of 4-aminobiphenyl in BALB/cStCrlfC3Hf/Nctr mice. Eur J Cancer Clin Oncol 21:865–
873.
Schmalix WA, Maser H, Kiefer F, Reen R, Wiebel FJ, Gonzalez F, Seidel A, Glatt H, Greim H,
and Doehmer J (1993) Stable expression of human cytochrome P450 1A1 cDNA in V79
Chinese hamster cells and metabolic activation of benzo[a]pyrene. Eur J Pharmacol 248:
251–261.
Shertzer HG, Dalton TP, Talaska G, and Nebert DW (2002) Decrease in 4-aminobiphenylinduced methemoglobinemia in Cyp1a2(⫺/⫺) knockout mice. Toxicol Appl Pharmacol 181:
32–37.
Smolen TN, Brewer JA, and Weber WW (1993) Testosterone modulation of N-acetylation in
mouse kidney. J Pharmacol Exp Ther 264:854 – 858.
Stabbert R, Schafer KH, Biefel C, and Rustemeier K (2003) Analysis of aromatic amines in
cigarette smoke. Rapid Commun Mass Spectrom 17:2125–2132.
Sugamori KS, Wong S, Gaedigk A, Yu V, Abramovici H, Rozmahel R, and Grant DM (2003)
Generation and functional characterization of arylamine N-acetyltransferase Nat1/Nat2 double-knockout mice. Mol Pharmacol 64:170 –179.
Tsuneoka Y, Dalton TP, Miller ML, Clay CD, Shertzer HG, Talaska G, Medvedovic M, and
Nebert DW (2003) 4-aminobiphenyl-induced liver and urinary bladder DNA adduct formation
in Cyp1a2(⫺/⫺) and Cyp1a2(⫹/⫹) mice. J Natl Cancer Inst 95:1227–1237.
Turesky RJ, Freeman JP, Holland RD, Nestorick DM, Miller DW, Ratnasinghe DL, and
Kadlubar FF (2003) Identification of aminobiphenyl derivatives in commercial hair dyes.
Chem Res Toxicol 16:1162–1173.
Uno S, Dalton TP, Derkenne S, Curran CP, Miller ML, Shertzer HG, and Nebert DW (2004) Oral
exposure to benzo[a]pyrene in the mouse: detoxication by inducible cytochrome P450 is more
important than metabolic activation. Mol Pharmacol 65:1225–1237.
Weisburger J (2002) Comments on the history and importance of aromatic and heterocyclic
amines in public health. Mutat Res 506 –507(C):9 –20.
Address correspondence to: Dr. Denis M. Grant, Department of Pharmacology, University of Toronto, Medical Sciences Building, 1 King’s College Circle,
Toronto, Ontario M5S 1A8, Canada. E-mail: [email protected]
Downloaded from dmd.aspetjournals.org at ASPET Journals on May 9, 2016
DNA adduct formation, then N-acetylation per se may not greatly
influence ABP-DNA adduct formation. In contrast, AF-DNA adduct
formation may be more greatly influenced by N-acetylation, since
clearance in the Nat1/2(⫺/⫺) mice exhibited a greater dependence on
acetylation. The prolonged retention of the chemical may allow for a
potentially increased toxic response, as shown with the slower clearance of benzo[a]pyrene in Cyp1a1(⫺/⫺) mice upon its oral administration and the formation of higher DNA adducts (Uno et al., 2004).
Nevertheless, the situation is not that simple; localized O-acetylation within tissues, such as the bladder, that are particularly susceptible to arylamine-induced carcinogenesis may play a more crucial
role in shaping toxic outcomes upon arylamine exposure. Although
PhIP does not appear to be N-acetylated, studies indicate that the
N-OH-PhIP metabolite can be subjected to O-acetylation (Fretland et
al., 2003). In addition, other enzymes such as sulfotransferases can
also participate in the bioactivation of some of these compounds
(Fretland et al., 2003), and thus, a multitude of enzyme systems can
influence whether or not toxic endpoints occur. Indeed, sulfation has
been suggested to be a primary route of bioactivation of the Nhydroxy metabolites of AF and AAF in the mouse (Lai et al., 1987;
Miller, 1994). Insofar as NATs can also contribute to either outcome,
the clearance of procarcinogens by this pathway may influence the
extent or balance of such other potential bioactivation reactions.
Tissue DNA adduct studies after arylamine exposure in our knockout
animal model and tumor bioassays will be of fundamental importance
in addressing whether NATs play a more important role in facilitating
or preventing toxic endpoints.