[CANCER RESEARCH 43, 3720-3724,
August 1983]
Kinetics of N- and C-Hydroxylations of 2-Acetylaminofluorene
Sprague-Dawley
in Male
Rat Liver Microsomes: Implications for Carcinogenesis
Michael E. McManus,1 Rodney F. Minchin, Nancy Sanderson, Peter J. Wirth, and Snorri S. Thorgeirsson
Laboratories of Carcinogen Metabolism [M. E. M., N. S., P. J. W., S. S. T.] and Experimental Therapeutics and Metabolism [R. F. M.], National Cancer Institute, Bethesda,
Maryland 20205
ABSTRACT
The metabolism of 2-acetylaminofluorene (AAF) has been stud
ied in male Sprague-Dawley rat liver microsomes over a concen
tration range of 0.02 to 300 U.M,and kinetic parameters have
been determined for five oxidative pathways. The /V-hydroxyla-
gens, considerable effort has been directed towards identifying
those isoenzymes of the cytochrome P-450 system that catalyze
these reactions (4, 9, 20).
The hepatocarcinogen AAF2 has been used extensively by our
laboratory (19) as well as others (11, 12, 21) to study mecha
nisms of metabolic activation and detoxification. The cytochrome
tion of AAF was best described by a single enzyme system with
P-450-mediated metabolism of AAF involves oxidation at both
a mean «„,
of 0.033 UM and a mean Vâ„¢*of 3.63 pmol/mg/min.
nitrogen and carbon atoms; the former leads to metabolic acti
Pretreatment of animals with 2,3,7,8-tetrachlorodibenzo-p-dioxin
vation while hydroxylation at positions 1, 3, 5, 7, and 9 on the
(TCDD) caused a marked induction of W-hydroxylase activity
fluorene ring are considered detoxification pathways (11,12,19,
while phénobarbital had no effect. Biphasic kinetics for the 7- 21). Experiments with purified rabbit hepatic cytochrome P-450
hydroxylation of AAF were observed in both control and TCDD(5) and rodent liver microsomes (19, 20) indicate that several
and phenobarbital-induced
microsomes. The high-affinity Km
isoenzymes may be involved in the metabolism of AAF. The
[0.051 ±0.015 (S.E.) MM;n = 3] in control microsomes was 3 relative importance of pathways leading to either metabolic ac
orders of magnitude lower than the low-affinity K,„
(103 ±16 /¿M;
tivation or detoxification of AAF will depend on the Kâ„¢values of
n = 3) indicating that each isoenzyme predominated at vastly
each isoenzyme involved. In human hepatic microsomal prepa
different substrate concentrations. The mean Vm«values for the
rations, it was recently shown that the relative quantity of each
low- and high-affinity enzymes were 3.5 and 1351 pmol/mg/min,
metabolite of AAF produced and the balance between metabolic
respectively. TCDD pretreatment markedly induced the activity
activation (W-hydroxylation) and detoxification (C-hydroxylation)
of the low-capacity enzyme and reduced the activity of the highvaried markedly with substrate concentration (10).
capacity enzyme. Phénobarbitalcaused a significant induction of
In the present study, we have investigated the kinetics of AAF
both enzyme pathways. Biphasic kinetics were also observed
metabolism in rat liver microsomes and report kinetic parameters
for the 5-, 3-, and 1-hydroxylations of AAF in control and phe
for 5 of the major oxidative pathways involved. In the past,
nobarbital-induced microsomes, but in TCDD-pretreated micro
analytical methods used to quantitate AAF metabolism used
somes only 1-hydroxylation exhibited biphasic kinetics. TCDD
thin-layer or paper chromatography and consequently lacked the
caused a marked induction of these metabolic pathways while
sensitivity necessary to accurately define the velocity curves for
each metabolite. Recently, high-pressure liquid chromatography
phénobarbitalhad no effect. Nonclassical kinetics were observed
for the 9-hydroxylation of AAF, and at high substrate concentra
(16,18) has allowed simultaneous determination of the 6 major
tions detoxification via this pathway and 7-hydroxylation predom
oxidative metabolites with sufficient accuracy that Michaelisinated. However, at low concentrations, metabolic activation of
Menten parameters can be computed although, as reported
AAF via W-hydroxylation was a major pathway. These data
herein, some sensitivity problems still exist. The significance of
indicate that multiple forms of cytochrome P-450 are involved in the kinetic parameters for the oxidative metabolism of AAF in
relation to multiple forms of cytochrome P-450 and the modulat
AAF metabolism and that the balance between metabolic acti
vation and detoxification of this substrate is dependent on both
ing effect of inducers on AAF metabolism and carcinogenicity
concentration and previous exposure to inducers.
are discussed.
INTRODUCTION
A considerable body of data indicate that differences in meta
bolic processing of chemical carcinogens are critical in determin
ing both species and organ sensitivity to individual compounds
(4, 11, 12, 15, 21). Therefore, characterization of pathways
involved in carcinogen metabolism as well as definition of the
regulatory control of these processes appear essential for a
comprehensive description of the carcinogenic process. Since
cytochrome P-450-dependent oxidation is important in both met
abolic activation and detoxification of most chemical carcino1To whom reprint requests should be addressed at: National Cancer Institute,
Building 37, Room 3B27, Bethesda, Md. 20205.
Received December 16,1982; accepted April 12,1983.
3720
MATERIALS AND METHODS
Chemicals. Randomly labeled [3H]AAF (18 Ci/mmol) was purchased
from Moravek Biochemicals (Brea, Calif.) and was purified to greater
than 98% purity by high-pressure liquid chromatography (18). Unlabeled
AAF and 2-aminofluorene were obtained from Eastman Organic Chemi
cals
from
was
was
(Rochester, N. Y.), and NADPH and sodium phénobarbital were
Sigma Chemical Co. (St. Louis, Mo.). Desferrioxamine mesylate
purchased from Ciba Pharmaceuticals (Summit, N. J.), and TCOD
from Dow Chemical Co. (Midland, Mich.). Authentic N-OH-AAF and
2The abbreviations used are: AAF, 2-acetylaminofluorene; TCDD, 2,3,7,8tetrachlorodibenzo-p-dioxm;
N-OH-AAF, N-hydroxy-2-acetylaminofluorene:
1-OHAAF, 1-hydroxy-2-acetylaminofluorene;
3-OH-AAF, 3-hydroxy-2-acetylammofluorene; 5-OH-AAF. 5-hydroxy-2-acetylaminofluorene;
7-OH-AAF. 7-hydroxy-2-acetylaminofluorene; 9-OH-AAF, 9-hydroxy-2-acetylaminofluorene.
CANCER
RESEARCH
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VOL. 43
MF Metabolism in Rat Liver Microsomes
1-OH-AAF were generously donated by Dr. Elizabeth Weisburger, Na
tional Cancer Institute, and 3-OH-AAF, 5-OH-AAF, 7-OH-AAF, 9-OHAAF, and 2-acetylaminofluorene-9-one
were prepared as described pre
viously (18). All other chemicals used were of analytical reagent grade.
Animals. Male Sprague-Dawley rats (Taconic Farms, Germantown,
N. Y.), weighing 180 to 220 g, were provided with food and water ad
libitum. TCDD, dissolved in com oil, was administered i.p. as a single
dose (50 /¿g/kg)48 hr prior to sacrifice. Sodium phénobarbital (75 mg/
kg) dissolved in 0.9% NaCI solution was administered i.p. daily for 3
days prior to sacrifice.
Preparation of Microsomes. Animals were killed by cervical disloca
tion, and their livers were removed, minced in ice-cold 0.2 M potassium
phosphate containing 0.15 M potassium chloride (pH 7.3), and then
homogenized with 4 volumes of this isotonic buffer with a Polytron
homogenizer (Model PT10-35; Kinematic, Switzerland). All tissue manip
ulations were carried out at 0-4°. The homogenates were centrifuged
at 9000 x g for 20 min in a Sorvall RC2-B centrifuge to remove nuclei,
mitochondria, and cell debris. The supernatant fraction was then centri
fuged at 105,000 x g for 60 min in a Beckman Model L5-50 ultracentrifuge to obtain the microsomal pellet. Microsomes were washed once
with buffer and resedimented by centrifugation at 105,000 x g for 60
min. The final washed microsomal pellet was resuspended in 0.2 M
potassium phosphate buffer (pH 7.3) containing 30% (v/v) glycerol, using
a glass homogenizer. Samples were either used immediately or stored
at -80° in 0.5-ml aliquots at a protein concentration of 8 to 15 mg/ml.
Control experiments showed no significant difference between the re
sults obtained using either fresh or stored microsomes. Microsomal
protein concentrations were determined by the method of Uowry ef al.
(8) with crystalline bovine serum albumin as a standard.
Incubation Mixture. A standard 1-ml incubation mixture contained
0.07 to 0.5 mg microsomal protein, 100 mM sodium fluoride, 83 HIM
phosphate buffer (pH 7.4), 1.8 mw NADPH, and 0.02 to 300 ^M [3H]AAF (3.6 x 107 to 1.5 x 105 dpm/nmol) dissolved in 8 p\ dimethyl
sulfoxide. NADPH was omitted from blanks and replaced by an equal
volume of buffer. All reactions were commenced by the addition of
microsomes and carried out under air at 37°in a metabolic shaker for 5
min. These conditions gave reaction rates that were zero order with
respect to cofactor and linear with respect to protein concentration and
time. Reactions were stopped by the addition of 1 ml sodium acetate (1
M; pH 6.5) and 5 ml of cold ether. Authentic standards of AAF and its
metabolites were added to the sodium acetate solutions as carriers and
to visualize absorbance peaks during chromatography. After 2 ether
extractions, the pooled extracts were evaporated to dryness under
nitrogen, and residues were dissolved in 0.1 ml of methanol for Chro
matographie analysis.
Chromatography. AAF and its metabolites were determined according
to the method of Smith and Thorgeirsson (18). Briefly, 10 n\ of the
reconstituted sample were injected onto a DuPont Zorbax C8 (4.6 mm x
15 cm) column. A flow rate of 1.5 ml/min was used, and absorbance
was monitored at 254 nm. The initial solvent was 28% isopropyl alcohol:72% 0.01 M acetic acid; both solutions contained 0.01% desferrioxamine mesylate to prevent chemisorption of N-OH-AAF and to provide
of the squared reciprocal of the observed data was used, and conver
gence was confirmed by initiating the iterative process from at least 3
independent initial parameter estimates. Randomness of residuals was
analyzed by the run sequence method (3), and models were compared
by the f test of Boxenbaum ef a/. (2). All data are expressed as mean ±
S.E. Standard errors of the parameters estimated by nonlinear regression
analysis were computed as described elsewhere (6).
RESULTS
The apparent kinetic parameters for 5 oxidative pathways of
AAF metabolism were determined in control rat liver microsomes.
The data for the A/-hydroxylation of AAF from all animals studied
was best described by a single enzyme system (Chart 1d). The
Michaelis-Menten parameters are summarized in Table 1. A
mean «„,
of 0.033 ±0.009 MM (range, 0.018 to 0.061) and a
mean Vm«of 3.63 ±1.59 pmol/mg protein/min (range, 0.95 to
8.02) were computed from the data. At substrate concentrations
less than 0.15 /IM, N-OH-AAF accounted for greater than 20%
of the metabolites formed. However, as the concentration of
substrate was increased, the contribution of W-hydroxylation
decreased markedly and accounted for less than 3% of the total
products formed at AAF concentrations greater than 10 /ÕM.
Hydroxylation of AAF at position 7 on the fluorene ring con
stituted a major metabolic pathway over the entire concentration
range studied. The data exhibited biphasic kinetics indicating the
presence of at least 2 isoenzymes of cytochrome P-450 respon
sible for the production of 7-OH-AAF. As shown in Chart 16, a
2-enzyme system best described the data. The smaller Km(0.051
±0.015 /¿M)
was 3 orders of magnitude lower than the larger Km
(103 ±16 MM),indicating that each isoenzyme of cytochrome P450 predominated at vastly different substrate concentrations
(Table 1). The first enzyme was readily saturated (V™«,
= 3.51
±1.36 pmol/mg protein/min), whereas the second had a meta
bolic capacity greater than 1300 pmol per mg protein per min.
The results indicate that the 7-hydroxylation of AAF is defined
by a high-affinity low-capacity enzyme and a low-affinity highcapacity enzyme.
The capacity of microsomes from control animals to 5-, 3-,
and 1-hydroxylate AAF was relatively low (Table 1). Chart 1, C
to E show that all 3 metabolites exhibited biphasic kinetics.
However, at substrate concentrations greater than 10 UM, the
sensitivity of the radiometrie assay precluded an accurate deter-
40°0
the best resolution of AAF and 8 of its metabolites (18). Instrumentation
consisted of an Altex Model 322 high-pressure liquid Chromatograph, 2
Altex Model 110A pumps, an Altex Model 420 microprocessor, a Gilson
Model 111 LC detector, and an Altex Model 2550 recorder.
Kinetic Analysis. Data were fitted stepwise to the general model
i
UKE
21Bk__500
V^S
iv "*"u)
_Lc\
(^
+ 1 <Fm
where v is velocity, S is substrate concentration, V™«is maximum
velocity, Km is Michaelis-Menten constant, and n is number of catalytic
sites.
Estimates of the model parameters and their respective variances
were determined by weighted nonlinear least-squares regression analysis
using a Levenberg-Marquardt algorithm [MLAB (6)]. A weighting factor
>
12.5
3.5
0
2.25
4.5
1,100
V {pmol/mg protein/min)
Chart 1. Eadie-Scatchardptots for the N-hydroxylation (A), 7-hydroxylation (B),
5-hydroxylatton (C), 3-hydroxylation (D), 1-hydroxylatton (£),and 9-hydroxylaaon
(F) of AAF by control rat liver microsomes.
AUGUST 1983
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3721
M.E. McManusetal.
Tabtel
Michaelis Menten parameters for oxidative pathways of AAF metabolism
Microsomes from 3 animals were pooled for the determination of AAF metabolism following TCDD and phénobarbital
pretreatments. The standard error of the estimated parameters was computed as described in "Materials and Methods."
hydrox-ylationsN-OH-AAF7-OH-AAF5-OH-AAF3-OH-AAF1-OH-AAFPretreatmentNoneTCDDPhénobarbitalNoneTCDDPhénobarbitalNoneTCDDPhénobarbitalNoneTCDD
AAF
(pmol/mg protein/
(pmol/mg
protein/min)3.63
min)1351
0a0.20±0.01
±0.020.20
±0.020.051
1.5972±
41.26±
±
0.143.51
0.0150.36
±
±0.040.38
±0.120.024
1.36543±
±5220.3
5.61.29±
±
0.0040.94
±
±0.080.37
±0.020.041
0.45695
±382.27
0.080.51
±
±0.0141.05
±0.120.42
±0.120.036
0.15631±
±403.84
1.030.36
±
0.0030.034
±
0.090.16
±
±0.04V™.,
8 Control values, mean ±S.E. from 4 animals.
" Average of 2 determinations.
0 Sensitivity of the assay precluded an accurate
1652
+
±76628
±39212
1402692
±
4538.03"CCCCCC
±
±525.13"CCCCCCV™«
0.1818.8
±
4.40.56
±
± 0.12K«(ft»)103
determination
of the parameters
defining the second enzyme
component.
mination of the metabolites produced. Consequently, saturation
of the high-capacity enzyme system could not be achieved,
preventing the kinetic parameters from being defined on most
occasions. The parameters describing the low-capacity system
are summarized in Table 1. In general, the Km and Vm«values
of the low-capacity enzymes responsible for 5-, 3-, and 1hydroxylations were very similar and indicated that these en
zymes were saturated at substrate concentrations greater than
1 UM. The high-capacity enzymes, while not defined in the
present study, contributed very little to the metabolism of AAF.
At substrate concentrations exceeding 10 //M, 5-, 3-, and 1hydroxylation accounted for less than 5% of the total products
formed.
Formation of 9-OH-AAF represented a major metabolic path
way comparable with 7-hydroxylation. Furthermore, at higher
substrate concentrations, 9-OH-AAF accounted for more than
40% of the total metabolites. An Eadie-Scatchard plot of the
data indicated that Michaelis-Menten kinetics were not adequate
for describing the rate of 9-OH-AAF formation (Chart 1F).
The effects of prior treatment of animals with TCDD and
phénobarbitalon the kinetic parameters for the oxidative metab
olism of AAF are shown in Table 1. In microsomes from rats
pretreated with TCDD, marked induction of the minor pathways
(5-, 3-, and 1-hydroxylations) was seen. The maximum velocity
for the formation of 5-OH-AAF and 3-OH-AAF was increased
>500-fold while that for 1-hydroxylation increased 50-fold. The
5- and 3-hydroxylations were best described by a single enzyme
system as opposed to control microsomes where they were
distinctly biphasic. For the 1-hydroxylation of AAF, biphasic
kinetics were apparent in both TCDD-pretreated microsomes
and in controls. Phénobarbital pretreatment had little effect on
the Vmaxof 5- and 1-hydroxylations of AAF and caused a 7.5fold increase in the Vâ„¢,of the 3-hydroxylation of AAF (Table 1).
Except for the 1-hydroxylation of AAF, both inducers caused a
significant increase (>6-fold) in the Km for each pathway; the
extent of the change in Km values was similar for both TCDD
3722
and phénobarbital. Biphasic kinetics were still evident after phé
nobarbital pretreatment.
Both TCDD and phénobarbitalpretreatment altered the kinet
ics of the 7-hydroxylation of AAF. However, the observed
changes were inducer dependent. Phénobarbitalcaused a sig
nificant induction of both isoenzymes responsible for the forma
tion of 7-OH-AAF. The Km for the low-capacity enzyme compo
nent was increased 7-fold whereas little change was seen in the
Kmof the high-capacity component. In contrast, TCDD markedly
induced the low-capacity enzyme and approximately halved the
metabolic capacity of the high-capacity enzyme. TCDD caused
a similar increase in the Km of the low-capacity component as
that induced by phénobarbital.
A/-Hydroxylation of AAF was markedly induced by TCDD but
not by phénobarbital (Table 1). Both inducers caused a 7-fold
increase in KH1and apparently neither produced a second enzyme
component capable of A/-hydroxylating AAF.
DISCUSSION
AAF has been used extensively over the last 2 decades to
study the mechanisms of chemical carcinogenesis and mutagenesis (11, 12, 19-21). Although considerable data exist on
AAF, we still lack precise information on the oxidative pathways
involved in its metabolism. In this study, we report for the first
time kinetic parameters for 5 of the 6 major oxidative pathways
involved in the metabolic disposition of AAF in the rat.
A/-Hydroxylation is considered the first and obligatory step in
the bioactivation of AAF and other aromatic amines and amides
to their ultimate carcinogenic or mutagenic forms (11, 12, 20,
21). In both control and induced rat liver microsomes, this
pathway was best described by a single high-affinity low-capacity
enzyme system (Table 1; Chart 1/4). The Km reported in the
present study for untreated male Sprague-Dawley rats (0.033 ±
0.010 /¿M)
is an order of magnitude lower than the Kmof 0.53 /IM
reported by Razzouk et al. (15) who used male Wistar rats.
CANCER
RESEARCH
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VOL. 43
AAF Metabolism in Rat Liver Microsomes
While both phénobarbital and TCDD increased the Km for this
of the above authors to detect a second enzyme component
reaction, only TCDD increased the Vâ„¢,.This is consistent with
may be due to the limited substrate concentration range used.
The 5- and 3-hydroxylations of AAF, like the W-hydroxylation,
studies using inbred strains of mice which have shown that the
/V-hydroxylation of AAF is controlled by the Ah receptor and is were substantially induced by TCDD pretreatment. In contrast,
associated with cytochrome P-448 (19). In addition to TCDD,
phénobarbital had little effect on the metabolic capacities of
other polycyclic hydrocarbons have been shown to induce Nthese pathways.
Except for the 1-hydroxylation of AAF, both inducers produced
hydroxylation of AAF, and the 20-fold induction observed in this
a consistent increase (>6-fold) in the Kmvalues for the oxidative
study is approximately one-half of that observed after benzanthracene induction (14).
pathways examined. The markedly different Km values seen in
The 7-hydroxylation of AAF in both control and induced rat
TCDD- and phenobarbital-pretreated microsomes compared to
liver microsomes exhibited biphasic kinetics (Table 1; Chart 1S).
control microsomes may reflect a change in the structure of the
Since multiple forms of cytochrome P-450 have been demon
cytochrome protein. Kinetic analysis of metabolic rates can reveal
strated in rat liver microsomes (9), it seems likely that the 2 «„,the presence of several different components only if the kinetic
parameters (K„,
and V™«)
are sufficiently different. Thus, when
values for this reaction represent the involvement of at least 2
isoenzymes of cytochrome P-450. In addition to multiple forms
several distinct isoenzymes are responsible for the metabolism
of cytochrome P-450 biphasic kinetic curves may also be indic
of a substrate but exhibit similar kinetics, the activity of each
isoenzyme will be "averaged" into a single component. In the
ative of multiple sites on a single isoenzyme or allosteric inter
present study, biphasic kinetics are indicative of at least 2 distinct
actions between the enzyme and substrate. To our knowledge,
isoenzymes. However, each component may be a composite of
no evidence is available supporting either of the latter mecha
several forms of cytochrome P-450. The change in Kmseen after
nisms. Johnson et al. (5) have previously shown that 4 highly
purified forms of rabbit liver cytochrome P-450 differ with respect
either TCDD or phénobarbital treatment may reflect selective
induction of an isoenzyme present only to a minor extent in
to rate and site specificity of AAF oxidation. Form 4, the major
cytochrome P-450 isoenzyme induced by TCDD in adult rabbit
control microsomes. This phenomena may also reflect product
liver, is the only form to catalyze AAF A/-hydroxylation. This form
inhibition, as Razzouk ef al. (15) have previously shown that 1OH-AAF, 3-OH-AAF, and 5-OH-AAF competitively inhibit the Nalso catalyzed the 7-, 5-, 3-, and 1-hydroxylations of AAF.
However, Form 6, the major isoenzyme of cytochrome P-450
hydroxylation of AAF.
induced by TCDD in neonatal rabbit liver, and Form 3, a consti
Oxidation of AAF at position 9 represents a major detoxifica
tion pathway in rats. Eadie-Scatchard plots of the rate of 9-OHtutive form of the cytochrome, both catalyze hydroxylation of
AAF exclusively in position 7. Form 2, the major phenobarbitalAAF formation exhibited nonnegative slopes, indicating that the
induced cytochrome P-450 isoenzyme in rabbit liver, exhibited
assumptions made with Michaelis-Menten kinetics are inade
no catalytic activity with AAF as substrate. The fact that phé quate for this pathway. Similar kinetics has been observed in
nobarbital and TCDD pretreatment have a differential effect on
hepatic microsomes from humans (10); however, the significance
the 7-hydroxylation of AAF in rat liver microsomes provides
of these observations are presently unknown.
further support for the involvement of multiple forms of cyto
Simulations using the derived kinetic constants from control
chrome P-450 in this pathway. TCDD pretreatment increased
and induced microsomes have been carried out to demonstrate
the activity of the low-capacity enzyme 240-fold but decreased
the relative contributions of each pathway to the total metabolism
the activity of the high-capacity enzyme approximately 2-fold
of AAF (Chart 2). Only substrate concentrations up to 10 UM
(Table 1). The overall metabolic capacity of TCDD-pretreated
were included in the simulations since it was assumed that
environmental exposure to and most bioassay protocols in carmicrosomes (Vm«,+ Vmax2)was not significantly different from
that of controls. Phénobarbital,on the other hand, increased the
cinogenicity studies with aromatic amides would not produce
metabolic capacity of the low- and high-capacity enzymes 6- and
significantly higher levels in vivo. Simulations based on data from
2.0-fold, respectively. It would appear that the forms of cyto
control animals clearly demonstrate that A/-hydroxylation of AAF
chrome P-450 induced by phénobarbitalin rat liver microsomes
is a major metabolic pathway at low substrate concentrations
have different catalytic properties to the major isoenzyme of
(Chart 2A). However, at high substrate concentrations, detoxifi
cytochrome P-450 that is inducible by phénobarbital in rabbit
cation of AAF via 7- and 9-hydroxylation are the major metabolic
pathways and N-OH-AAF comprises less than 3% of the total
liver (Form 2).
metabolites. Thus, because of the low Kmfor the A/-hydroxylation
Biphasic kinetics were also observed for the 5-, 3-, and 1hydroxylations of AAF in control and phenobarbital-induced mi
of AAF, this pathway is readily saturable, and at increasing
crosomes but, in TCDD-pretreated microsomes, only 1-hydrox
substrate concentrations the amount of the dose undergoing
ylation was biphasic. The sensitivity of the assay often precluded
metabolic activation is not increased.
Although TCDD significantly increases the rate of AAF A/an accurate determination of the second enzyme component. In
agreement with previous animal studies, these metabolities were
hydroxylation, the relative contribution of this pathway is de
products of low-capacity pathways (7,17-19, 21). However, in
creased at low substrate concentrations due to much larger
human microsomes, 1-hydroxylation was a major pathway ac
increases in 3- and 5-hydroxylation of AAF (Chart 2B). For
counting for up to 50% of the metabolites formed (10). Batardyexample, A/-hydroxylation comprises about 5% of the total me
Gregoire ef al. (1) have previously reported the kinetic constants
for the 5- and 7-hydroxylation of AAF by microsomes from male
Wistar rats and hamsters. In their study, only a single enzyme
component for each reaction was seen over a substrate concen
tration range of 0.25 to 5 /IM. In the present study, the concen
tration of AAF was varied from 0.02 to 300 ¿tM,
and the inability
tabolites at 1 UM AAF after TCDD treatment as compared to
17% in control microsomes (Chart 2, A and B). Similarly, phé
nobarbital pretreatment decreased the percentage of the dose
going via A/-hydroxylation at low substrate concentrations and
increase the percentage of 7-OH-AAF formed at all concentra
tions of AAF (Chart 2C). These data are consistent with the
AUGUST 1983
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3723
M. E. McMan us et al.
701—
minations of AAF metabolism can be misleading as to the relative
importance of each oxidative pathway. Furthermore, because of
the sensitivity of the radiometrie high-pressure liquid chromatog-
7-OH
raphy assay used herein and in several other studies, relatively
high AAF concentrations (>10 UM) may incorrectly indicate that
certain oxidative products cannot be produced.
9-OH
REFERENCES
5-OH
3-OH
/ X
9-OH
N-OH
1-OH
7-OH
9-OH
15
5-OH
+
............
••'^Tr.'.-.Ttar^-.na
10
AAF CONCENTRATION I
Chart 2. Simulations of trie metabolism of AAF based on kinetic constants
determined in control microsomes (A) and TCDD (B)- and phénobarbital (C)pretreated microsomes.
observed reduction in AAF carcinogenicity following pretreat
ment of rats with phénobarbital(13).
In summary, the present study has reported the kinetic con
stants for 5 oxidative pathways involved in AAF metabolism in
rat liver microsomes and found nonclassical kinetics for another.
The biphasic kinetics observed for the 7-, 5-, 3-, and 1-hydroxylations of AAF implicate at least 2 forms of cytochrome P-450
being involved in the metabolism of this substrate. Furthermore,
the relative importance of the metabolic activation and detoxifi
cation pathways of AAF is dependent both on substrate concen
tration and previous exposure to inducers. Single-point deter
3724
1. Batardy-Gregoire, M.. Razzouk, C., Evrard, E., and Roberfroid. M. Quantitative
analysis of the ring-hydroxylated metabolites of 2-fluorenylacetamide by gasliquid chromatography. Anal. Biochem., 122:199-205,1982.
2. Boxenbaum, H. G., Riegelman. S., and Elashoff, R. M. Statistical estimations
in pharmacokinetics. J. Pharmacokinet. Biopharm., 2:123-148,1974.
3. Draper, N. R . and Smith, H. Applied Regression Analysis, pp. 95-99. Sydney:
John Wiley & Sons, Inc., 1966.
4. Gelboin. H. V. Benzo|aIpyrene metabolism, activation and carcinogenesis: rote
and regulation of mixed-function oxidases and related enzymes. Physio!. Rev.,
60: 1107-1166,1980.
5. Johnson, E. F., Levitt, D. S., Multer-Eberhard, U., and Thorgeirsson, S. S.
Divergent pathways of carcinogen metabolism: metabolism of 2-acetylaminoftuorene by multiple forms of cytochrome P-450. Cancer Res., 40:4456-4460,
1980.
6. Knott, G. D. MLAB—a mathematical modeling tool. Comput. Programs
Biomed., 10: 271-280,1970.
7. Lotlikar, P. D., Enomoto, M., Miller, J. A., and Miller, E. C. Species variations
in the N- and nng-hydroxylation of 2-acetylaminofluorene and effects of 3methyteholanthrene pretreatment. Proc. Soc. Exp. Btol. Med., 725; 341-346,
1967.
8. Lowry, 0. H., Rosebrough, N. J., Fair, A. L, and Randall, R. J. Protein
measurements with the Folin phenol reagent. J. Biol. Chem., 793: 265-275,
1951.
9. Lu, A. Y. H., and West, S. B. Multiplicity of mammalian microsomal cytochrome
P«o.Pharmacol. Rev., 31: 277-295,1980.
10. McManus, M. E., Minchia R. F., Sanderson, N. S., Wirth, P. J., and Thorgeirs
son, S. S. Kinetic evidence for the involvement of multiple forms of human liver
cytochrome P<M)in the metabolism of acetylaminofluorene. Carcinogenesis
(Lond.), in press, 1983.
11. Miller, E. C. Some current perspectives on chemical carcinogenesis in humans
and experimental animals: presidential address. Cancer Res., 38:1479-1496,
1978.
12. Miller, J. A. Carcinogenesis by chemicals: an overview—G. H. A. Clowes
Memorial Lecture. Cancer Res., 30: 559-576,1970.
13. Peraino, C., Fry, R. J., and Staffeldt, E. Reduction and enhancement by
phénobarbitalof hepatocarcmogenesis induced in the rat by 2-acetylaminoflu
orene. Cancer Res., 37: 1506-1512,1971.
14. Razzouk, C., Batardy-Gregoire, M., and Roberfroid, M. Induction and modifi
cation of rat liver microsomal arylamide W-hydroxylase by various pretreatments. Mol. Pharmacol., 27: 449-457,1982.
15. Razzouk, C., Mercier, M., and Roberfroid, M. Induction, activation, and inhibi
tion of hamster and rat liver microsomal arylamide and arylamine W-hydroxyl
ase Cancer Res., 40: 96-102,1980.
16. Ryzewski, C. N., and Male|ka-Giganti. D. Systems for the separation of
metabolites of the carcinogen N-2-fluorenylacetamide
by high-performance
liquid chromatography. J. Chromatogr., 237: 447-456,1982.
17. Schut, H. A. J., and Thorgeirsson, S. S. In vitro metabolism and mutagenic
activation of 2-acetylaminofluorene and W-hydroxy-2-acetylaminofluorene
by
cotton rat subcellular fractions. Cancer Res., 38:2501-2507,1978.
18. Smith, C. L., and Thorgeirsson, S. S. An improved high-pressure liquid Chro
matographie assay for 2-acetylaminofluorene and eight of its metabolites. Anal.
Biochem.. 773: 62-67,1981.
19. Thorgeirsson, S. S., Glowinski, I. B., and McManus, M. E. Metabolism,
rnutagencity and carcinogenicity of aromatic amines. Rev. Biochem. Toxicol..
in press, 1983.
20. Thorgeirsson, S. S., and Neben. D. W. The Ah locus and the metabolism of
chemical carcinogens and other foreign compounds. Adv. Cancer Res., 25:
149-193,1977.
21. Weisburger, J. H., and Weisburger, E. K. Biochemical formation and pharma
cological, toxicological and pathological properties of hydroxylamines and
hydroxamic acids. Pharmacol. Rev., 25:1-66,1973.
CANCER RESEARCH
VOL. 43
Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1983 American Association for Cancer Research.
Kinetics of N- and C-Hydroxylations of 2-Acetylaminofluorene in
Male Sprague-Dawley Rat Liver Microsomes: Implications for
Carcinogenesis
Michael E. McManus, Rodney F. Minchin, Nancy Sanderson, et al.
Cancer Res 1983;43:3720-3724.
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