DMD Fast Forward. Published on June 30, 2006 as DOI: 10.1124/dmd.106.010819
DMD Fast
Forward.
Published
30,The
2006
as doi:10.1124/dmd.106.010819
This article
has not been
copyedited on
and June
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DMD # 10819
In Vivo and In Vitro Metabolism of Arylamine
Procarcinogens in Acetyltransferase-Deficient Mice
K.S. Sugamori, D. Brenneman, and D.M. Grant
-1Copyright 2006 by the American Society for Pharmacology and Experimental Therapeutics.
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Department of Pharmacology, Faculty of Medicine (KSS, DB, DMG), and
Leslie Dan Faculty of Pharmacy (DMG)
University of Toronto
Toronto, Canada M5S 1A8
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Running Title:
NAT function in genetically modified mice
Corresponding Author:
Denis M. Grant, Ph.D.
Department of Pharmacology
University of Toronto
Medical Sciences Building
1 King’s College Circle
Toronto, Ontario M5S 1A8
Telephone: 416-978-2723
Fax:
416-978-6395
Email:
[email protected]
13
Number of Tables:
1
Number of Figures:
5
Number of References:
42
Number of Words in Abstract:
250
Number of Words in Introduction:
751
Number of Words in Discussion:
1141
List of Abbreviations: ABP, 4-aminobiphenyl; AABP, N-acetyl-4-aminobiphenyl; AF, 2aminofluorene; AAF, N-acetyl-2-aminofluorene; NAT, N-acetyltransferase: PAS, paminosalicylic acid; APAS, N-acetyl-PAS; PhIP, 2-amino-1-methyl-6-phenyl[4,5-b]pyridine;
APhIP, N-acetyl-PhIP; SMZ, sulfamethazine; ASMZ, N-acetyl-SMZ
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Number of Text Pages:
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ABSTRACT
Arylamine N-acetyltransferases (NATs) catalyze the biotransformation of a number of aromatic
and heterocyclic amines, many of which are pro-carcinogenic 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 tissue-specific metabolism
acetylation on the metabolism of some classical pro-carcinogenic arylamines, we have used our
double knockout Nat1/2(-/-) 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(-/-) mice for 4-aminobiphenyl (ABP) and 2-aminofluorene (AF), while
significant levels were measured in all wild-type tissue cytosols tested indicating the widespread
metabolism of these agents. Nat1/2(-/-) mice displayed a variable response with respect to in
vivo pharmacokinetics. AF appeared to be most severely compromised with a 3-4-fold increased
area under the curve (AUC), while the clearance of ABP was found to be less dependent on Nacetylation with no difference in ABP-AUC between wild-type and knockout animals. 2-amino1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) 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 pro-carcinogenic agents is the
first step in attempting to correlate metabolism by NATs to toxic outcome prevention or
augmentation.
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by competing but genetically variable drug metabolizing enzymes. To investigate the effect of N-
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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 drug
metabolizing enzymes. In particular, the polymorphic arylamine N-acetyltransferases (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 Butcher et
carcinogenic arylamines into innocuous metabolites by N-acetylation or promote their metabolic
activation into DNA-binding electrophiles via O-acetylation (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
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al., 2002; Grant et al., 2000). Paradoxically, NATs can either facilitate the detoxification of
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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
Schieferstein et al., 1985), human tissue samples (Airoldi et al., 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 due to differences in sample size,
genotyping/phenotyping discordances and variable exposure situations.
While 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 PhIPinduced 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
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models (Flammang et al., 1992; Fletcher et al., 1998; Levy and Weber, 1992; Poirier et al., 1995;
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et al., 2004), despite in vitro studies which 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
the importance of acetylation on the metabolism and clearance of these compounds in the whole
animal. While 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.
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clearance of the pro-carcinogens ABP, AF and PhIP. This is a necessary step in dissecting out
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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-DLcarnitine, carnitine acetyltransferase, acetyl-CoA sodium salt, ABP, AF and AAF used for NAT
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 µl, each
consisting of 10 µl of ABP or AF solution (giving a final concentration of 0.1 mM), 20 µl of 0.5
mM acetyl-CoA (giving a final concentration of 0.1 mM), 20 µl of an acetyl-CoA regenerating
system composed of 5 mM acetyl-DL-carnitine and 1 U carnitine acetyltransferase per milliliter
of assay buffer (250 mM triethanolamine-HCl, 5 mM EDTA, 5 mM DTT, pH 7.5), and 50 µl of
diluted cytosol at a protein concentration of 1 µg/µl. Reactions were initiated with the addition of
the cytosol to the remaining pre-heated components, incubated for 10 min at 37° and terminated
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activity assays were acquired from Sigma-Aldrich Canada Ltd. (Oakville, ON, Canada). AABP
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by the addition of 10 µl of 15% perchloric acid. For PhIP, the reactions were initiated with
either 50 or 100 µg of cytosolic protein and were allowed to proceed for 60 min at 37° 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 LC2010A system (Shimadzu Scientific Instruments Inc., Columbia, MD) and a reverse-phase
Beckman Ultrasphere ODS 5 µm column (15 cm x 4.6 mm I.D.; Beckman Instruments,
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 DMSO was
administered by i.p. injection to 8- to 9-week-old age- and sex-matched Nat1/2(-/-) mice (n= 4)
and wild-type sibs (n=4). Blood samples were drawn from the saphenous vein using heparinized
microvettes (Sartstedt Inc., Montreal, QC, Canada) at four sequential timepoints and centrifuged
to separate plasma. The timepoints for AF plasma sampling were 2, 6, 22 and 24 hr for female
knockout mice, and 1, 2, 4 and 6 hr for female wild-type, male wild-type and male knockout
mice. ABP plasma sampling times were 2, 6, 22 and 24 hr for all genders and genotypes. PhIP
plasma sampling times were 30 min, 60 min, 90 min and 120 min for all animals. Plasma
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Fullerton, CA). A flow rate of 2 ml/min with a mobile phase consisting of 66% sodium
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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 values (AUC) 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.
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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 two-fold higher activity (p < 0.001) than
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 to ABP in the wild-type animals (Fig. 2A, C) and the lack of a
significant change in the ABP-AUC values in the knockout animals compared to wild-type
(Table 1). Knockout animals for both genders did display a complete absence of AABP (Fig. 2B,
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 ~3-4-fold higher AF-AUC values from the knockout animals compared to wild-type
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female kidney cytosol for both AF and ABP. No other tissue cytosols displayed a gender
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for both genders (Fig. 3, Fig. 5), although the mean AF-AUC value for wild-type females was
about 3-fold higher than wild-type males (Table 1, p< 0.05). Knockout animals displayed a
complete absence of N-acetylated metabolite in all plasma samples analyzed (Fig. 3B, D). The
small AUC values for AAF compared to 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. 4). AUC values for the parent PhIP were not significantly different
to either AF or ABP, however, indicates that PhIP is rapidly eliminated from plasma, presumably
by other pathways that may include redistribution into other tissue compartments or efficient
renal clearance.
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between wild-type and knockout animals (Table 1). The smaller AUC values for PhIP compared
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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 pro-carcinogens, 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
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 (PAS) 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 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 AFDNA 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 (Flammang et al., 1992; Levy and Weber, 1992; Poirier et al., 1995; Schieferstein et
al., 1985; Tsuneoka et al., 2003). Female mice, on the other hand, had higher liver DNA adduct
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susceptible to the formation of tumors upon ABP administration. No N-acetylation of PhIP
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levels (Chen et al., 2005; Levy and Weber, 1989; Poirier et al., 1995; Tsuneoka et al., 2003) and
a higher incidence of liver cancer (Poirier et al., 1995; Schieferstein et al., 1985).
In order 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,
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
been have higher hepatic AF-DNA adducts, is related to this difference in clearance.
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 to AF and ABP suggests that PhIP is rapidly cleared from
plasma. This would implicate either the involvement 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
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as determined by the 3-4 fold increase in AF-AUC values for the knockout animals compared to
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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 pro-carcinogens
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
DNA adduct formation then N-acetylation per se may not greatly influence ABP-DNA adduct
formation. On the other hand, AF-DNA adduct formation may be more greatly influenced by Nacetylation, 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).
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ABP, AF and PhIP display a differential dependence on N-acetylation for clearance. What effect
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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. While PhIP does not
appear to be N-acetylated, studies indicate that the N-OH-PhIP metabolite can be subjected to Oacetylation (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,
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 pro-carcinogens 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.
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sulfation has been suggested to be a primary route of bioactivation of the N-hydroxy metabolites
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ACKNOWLEDGEMENTS
We thank Roopam Singh and Simin Yazdanpanah for their excellent technical assistance.
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exposure to benzo[a]pyrene in the mouse: detoxication by inducible cytochrome P450 is
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FOOTNOTES
This research was supported by an operating grant from the National Cancer Institute of Canada,
with funding from the Canadian Cancer Society.
Reprint requests should be addressed to:
D.M. Grant, Ph.D.
Department of Pharmacology
University of Toronto
1 King’s College Circle
Toronto, Ontario M5S 1A8
[email protected]
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LEGENDS FOR FIGURES
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 in the 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
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 µM.
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
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0.02 nmol/min/mg for AAF, AABP and APhIP, respectively.
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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
the N-acetylated metabolite was 4 µM.
Fig. 4. 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
µM.
Fig. 5. Graphical representation of AUC values for knockout animals plotted as a percent of
wild-type AUC. Values for PAS and SMZ were taken from Sugamori et al., 2003.
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trapezoidal rule using the computer program Prism (GraphPad Software Inc.). Detection limit for
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TABLE 1
AUC values for ABP, AF and PhIP elimination
ABP
AcABP
AF
AcAF
PhIP
APhIP
Male +/+
219±72
5.8±0.9**
24.7±4.8*
2.3±0.9*
5.4±0.7
0
Male -/-
264±64
0
85.8±30.0
0
5.2±1.3
0
Female +/+
195±8
7.1±1.9**
71.7±36.2*
4.0±2.1*
6.3±0.8
0
Female -/-
190±30
0
314±124
0
7.3±1.2
0
AUC values (mmol min l-1, mean ± SD, n=4) were determine by the trapezoidal rule using Prism
software. ABP, AF or PhIP (50 mg/kg) was dosed by i.p. injection to age- and sex-matched wildtype and knockout mice. Four sequential blood samples were drawn and analyzed for parent and
metabolite compounds as described under Materials and Methods. *, significantly different from
knockout of same gender (p < 0.05) **, significantly different from knockout of same gender (p
< 0.001).
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Genotype