TOXICOLOGY AND APPLIED PHARMACOLOGY
ARTICLE NO.
141, 595–606 (1996)
0326
The Kinetics of Aflatoxin B1 Oxidation by Human cDNA-Expressed and
Human Liver Microsomal Cytochromes P450 1A2 and 3A4
EVAN P. GALLAGHER,* KENT L. KUNZE,† PATRICIA L. STAPLETON,*
AND
DAVID L. EATON*,1
Departments of *Environmental Health and †Medicinal Chemistry, University of Washington, Seattle, Washington 98195
Received April 18, 1996; accepted July 24, 1996
The Kinetics of Aflatoxin B1 Oxidation by Human cDNAExpressed and Human Liver Microsomal Cytochromes P450
1A2 and 3A4. GALLAGHER, E. P., KUNZE, K. L., STAPLETON,
P. L., AND EATON, D. L. (1996). Toxicol. Appl. Pharmacol. 141,
595 – 606.
The combined presence of CYP1A2 and 3A4, both of which
oxidize aflatoxin B1 (AFB1 ) to the reactive aflatoxin B1-8,9-epoxide
(AFBO) and to hydroxylated inactivation products aflatoxin M1
(AFM1 ) and aflatoxin Q1 (AFQ1 ), substantially complicates the
kinetic analysis of AFB1 oxidation in human liver microsomes. In
the present study, we examine the reaction kinetics of AFB1 oxidation in human liver microsomes (HLMs, N Å 3) and in human
CYP3A4 and CYP1A2 cDNA-expressed lymphoblastoid microsomes for the purpose of identifying the CYP isoform(s) responsible for AFB1 oxidation at low substrate concentrations approaching those potentially encountered in the diet. AFBO formation by cDNA-expressed human CYP1A2 followed Michaelis–
Menten kinetics (Km Å 41 mM, Vmax Å 2.63 nmol/min/nmol P450).
Furthermore, the portion of AFBO formed in HLMs which was
eliminated by furafylline, a specific mechanism-based inhibitor of
CYP1A2, also followed Michaelis–Menten kinetics (Km Å 32-47
mM, Vmax Å 0.36-0.69 nmol/min/nmol P450). The formation of
AFBO (activation product) and AFQ1 (detoxification product) in
cDNA-expressed human CYP3A4 microsomes was sigmoidal and
consistent with the kinetics of substrate activation. Accordingly,
application of a sigmoid Vmax model equivalent to the Hill equation
produced excellent fits to the cDNA-expressed CYP3A4 data and
also to the data from HLMs pretreated with furafylline to remove
CYP1A2. The Hill model predicted that two substrate binding
sites are involved in CYP3A4-mediated AFB1 catalysis and that
the average affinity of AFB1 for the two sites was 140–180 mM.
Vmax values for AFQ1 formation were 10-fold greater than those
for AFBO, and total substrate turnover to both was 67 nmol/min/
nmol CYP3A4. Using the derived kinetic parameters for CYP1A2
and 3A4 to model the in vitro rates of AFB activation at low
substrate concentrations, it was predicted that CYP1A2 contributes to over 95% of AFB activation in human liver microsomes
at 0.1 mM AFB. The important role of CYP1A2 in the in vitro
activation of AFB at low substrate concentrations was supported
1
To whom correspondence should be addressed at the Department of
Environmental Health, University of Washington, 4225 Roosevelt Way,
NE, Suite 100, Seattle, WA 98105-6099. Telephone: (206) 685-3785. Fax:
(206) 685-4696.
by DNA binding studies. AFB1 –DNA binding in control HLMs
(reflecting the contribution of CYP1A2 and CYP3A4) and furafylline-pretreated microsomes (reflecting the contribution of CYP3A4
only) catalyzed the binding of 1.71 and 0.085 pmol equivalents of
AFB1 to DNA, respectively, indicating that CYP1A2 was responsible for 95% of AFB1 –DNA adduct formation at 0.133 mM AFB.
These results demonstrate that CYP1A2 dominates the activation
of AFB in human liver microsomes in vitro at submicromolar
concentrations and support the hypothesis that CYP1A2 is the
predominant enzyme responsible for AFBO activation in human
liver in vivo at the relatively low dietary concentrations encountered in the human diet, even in high AFB exposure regions of
the world. However, because the actual concentrations of AFB in
liver in vivo following dietary exposures are uncertain, additional
studies in exposed human populations are needed. Quantitative
data on the relative rates of AFM1 and AFQ1 excretion (potential
biomarkers for CYP1A2 and 3A4 activity, respectively) in humans
would be useful to validate the actual contributions of these two
enzymes to AFB1 oxidation in vivo. q 1996 Academic Press, Inc.
The aflatoxins constitute a group of closely related difuranocoumarin mycotoxins which is an important public health
problem in regions where high heat and humidity favor the
mold growth and where food storage is inadequate. The
focus of this concern centers upon the carcinogenic effects
of aflatoxins, as there is substantial experimental animal
(Busby and Wogan, 1984; Roebuck and Maxuitenko, 1994;
Wogan, 1973) and human epidemiological (Groopman,
1994; Groopman et al., 1988; Hall and Wild, 1994) evidence
to provide a contributing link of these compounds to the
unusually high incidence of liver cancer in these areas. Aflatoxin B1 (AFB1 ) is the most potent of these mycotoxins and
requires microsomal cytochrome P450-mediated oxidation
at the 8,9 vinyl bond to produce a reactive intermediate,
aflatoxin B1-8,9-epoxide (AFBO), that accounts for its genotoxic properties. The critical initial lesion, 8,9-dihydro-8(N7-guanyl)-9-hydroxy-AFB1 , is formed in the reaction of
DNA with AFB1 exo-epoxide (Lin et al., 1977; Iyer et al.,
1994). Accordingly, much of the interspecies differences
with respect to susceptibility to AFB1 hepatocarcinogenesis
can be attributed to differences in AFB1 biotransformation
pathways (Eaton and Gallagher, 1994).
595
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Copyright q 1996 by Academic Press, Inc.
All rights of reproduction in any form reserved.
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GALLAGHER ET AL.
Previous studies from our laboratory (Gallagher et al.,
1994; Ramsdell et al., 1991) and others (Aoyama et al.,
1990; Forrester et al., 1990) have demonstrated that the
biotransformation of AFB1 in human liver is a complex
process that is controlled by multiple P450 enzymes exhibiting different kinetic characteristics. While at least five
different human liver CYP enzymes, including CYP1A2,
2A6, 2B7, 3A3 and 3A4, are capable of activating AFB1
to mutagenic metabolites (Aoyama et al., 1990), CYP1A2
and 3A4 appear to be the predominant catalysts of AFB1
epoxidation in human liver in vitro (Gallagher et al., 1994;
Raney et al., 1992) and in primary cultured human hepatocytes (Langouet et al., 1995). Paradoxically, human microsomal biotransformation of AFB1 also results in the production of water soluble metabolites aflatoxin Q1 (AFQ1 ) and
aflatoxin M1 (AFM1 ) which are primarily catalyzed by
CYP3A4 and CYP1A2, respectively (Gallagher et al.,
1994; Raney et al., 1992), and do not appear to share the
potent carcinogenic characteristics of AFBO. Relative to
CYP3A4, 1A2 produces a higher ratio of activation
(AFBO) to detoxification (AFM1) products (Gallagher et
al., 1994). However, there is some discrepency in the literature regarding the relative importance of CYP1A2 and 3A4
in human AFB1 metabolism, especially at the low substrate
concentrations that typically occur under in vivo conditions
(Forrester et al., 1990; Gallagher et al., 1994; Ramsdell et
al., 1991; Raney et al., 1992; Ueng et al., 1995). An estimated upper limit to concentrations of AFB1 in human liver
may be calculated from the highest estimate of the toxin
ingested (1 mg/week or 450 nmol day) divided by total
liver volume (1.8 liters), or roughly 0.25 mM (Groopman,
1994). This estimate assumes that all of the aflatoxin ingested is coincidentally found unchanged in the liver and
thus is likely to overestimate actual levels by 1 to 2 orders
of magnitude, even in highly exposed populations. In any
event, the in vivo target substrate concentrations are much
less than those which may be used in kinetic experiments
in vitro due to assay sensitivity limitations.
In the present study we have provided a detailed kinetic
analysis of AFB1 oxidation and the specific contributions of
CYP1A2 and 3A4 in the activation and detoxification of
AFB1 . In this study we used microsomes from lymphoblastoid cells that stably express either human CYP1A2 or 3A4
cDNA, and also human liver microsomes that contain multiple cytochromes P450, including CYP1A2 and 3A4. The
specific contribution of each of these forms was determined
by selectively and completely inhibiting 1A2 with furafylline. One goal was to ascertain the relative importance of
CYP3A4 and 1A2 in the formation of the highly toxic AFBO
through a determination of kinetic parameters Vmax and Km
for each enzyme. A second goal was to determine which
phase I metabolic products might be expected to represent
significant routes of detoxification. A kinetic approach offers
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the advantage of providing parameters which may be used
to estimate rates at the low concentrations of substrate which
occur in vivo as a consequence of dietary exposure to the
toxin.
MATERIALS AND METHODS
Chemicals. Furafylline was synthesized as described previously (Kunze
and Trager, 1993). AFB1 , AFM1 , aflatoxin G1 (AFG1 ), and AFQ1 , NADP/,
glucose 6-phosphate, glucose 6-phosphate dehydrogenase, reduced glutathione (GSH), and buffers were purchased from Sigma Chemical Co. (St.
Louis, MO). HPLC-grade solvents were purchased from J.T. Baker, Inc.
(Philipsburg, NJ). [3H]AFB1 was purchased from Moravek Biochemicals
(Brea, CA). All other solvents and salts were analytical reagent grade and
were obtained from commercial sources.
Human liver microsomal preparations. Human liver samples were obtained from three organ donors through the University of Washington Hospital (Seattle, WA) and stored at 0807C until microsomal preparation. The
age, gender, and drug histories of the donors are as follows: HLM 105,
male, age 21, no medication history; HLM 109, male, age 46, Inderal;
HLM 125, male, age 33, phenytoin and dexamethasone. The method of
preparation of the human liver microsomes used in this study has been
described elsewhere (Rettie et al., 1989). Microsomes were stored at 0807C
until use. Protein concentrations were determined using the bicinchoninic
acid method of Smith et al. (1985) with bovine serum albumin as a standard.
Lymphoblastoid microsomes expressing human CYP1A2 or 3A4 cDNA.
Lymphoblastoid microsomes expressing human CYP1A2 or 3A4 cDNAs
(10 mg microsomal protein/ml) and containing cytochrome P450 reductase/
cytochrome b5 were obtained from Gentest Inc. (Woburn, MA). The approximate cytochrome P450 content for the CYP1A2 microsomes was 34 pmol/
mg protein, whereas the approximate cytochrome P450 content for the
CYP3A4 microsomes was either 39 or 44 pmol/mg protein, depending
upon the lot. The CYP1A2 and 3A4 microsomes were prepared by cDNAmediated gene transfer into human B-lymphoblastoid AHH-1 TK { cell
lines (Crespi et al., 1991).
AFB1 oxidation assay. Microsomal oxidation of AFB1 was carried out
by the method described previously (Gallagher et al., 1994) with the following modification: AFB1 in DMSO (10 ml) was added to the reaction mixtures
to give final concentrations ranging from 0.5 to 512 mM as determined by
UV spectrophotometric analyses of stock solutions (Busby and Wogan,
1984). Parallel incubations for each concentration of AFB1 were conducted
in the absence of microsomal protein. Incubation mixtures included hepatic
cytosol from rats and BHA-treated mice (2:1) to quantitatively trap the
reactive AFB1 –epoxide as the GSH conjugate. This method does not separate the AFBO endo- and exo-stereoisomers (Gallagher et al., 1994). HPLC
analyses were conducted as described previously (Monroe and Eaton, 1987)
with detection of AFB1 metabolites carried out using simultaneous UV (365
nm) and fluorescence (excitation 365 nm, emission 425 nm) detection.
AFQ1 , AFM1 , and the AFBO–GSH conjugate were quantitated directly,
with AFG1 (10 mM) as an internal standard (Gallagher et al., 1994).
AFB1-DNA binding experiments. Incubation mixtures (250 ml total
volume) contained 250 mg calf thymus DNA, CYP source (150 pmol human
liver microsomal P450, 10.2 pmol CYP1A2 microsomes, or 11.7 pmol
CYP3A4 microsomes), and NADPH regenerating system (1.0 mM NADP/,
0.5 U/ml glucose 6-phosphate dehydrogenase, and 5.0 mM glucose 6-phosphate) in 0.1 M KPO4 , pH 7.6. After 10 min at 377C, the reactions were
initiated by the addition of [3H]AFB1 in DMSO. In Experiment 1, the CYP
source was HLM microsomes, and the specific activity of the [3H]AFB1
was 3685 mCi/mmol (0.133 mM AFB1 final concentration after dilution in
unlabeled AFB1 ). In the second DNA binding experiment, the CYP source
was CYP1A2 and 3A4 cDNA-expressed microsomes, and the specific activity of the [3H]AFB1 was 85 mCi/mmol (15.3 mM AFB1 final concentration
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597
after dilution in unlabeled AFB1 ). After 0 to 60 min, the reactions were
stopped by addition of 250 ml of chloroform/isoamyl alcohol/phenol (CIP,
25/24/1) and vortexing. After a brief centrifugation, the aqueous phase was
transferred and the DNA–protein interface was back-extracted with CIP,
recentrifuged, and combined with the aqueous fraction. The aqueous phase
was then further extracted three times with chloroform/isoamyl alcohol (24/
1) and vortexed. The aqueous phase containing DNA was precipitated with
2 vol of ice-cold ethanol and 0.1 vol 3M sodium acetate (pH 5.0) and
precipitated on ice. After centrifugation, the DNA was washed in 70%
ethanol, dried, and resuspended in 1 ml 10 mM Tris–HCl, pH 7.6, containing
1 mM EDTA. Purity and quantitation of DNA were verified spectrophotometrically by measuring at 260 and 280 nm. Bound radioactivity was determined in a second aliquot (800 ml) of DNA by scintillation counting after
addition of 4.5 ml of Ecolum counting fluid.
Inhibition of microsomal CYP1A2 activities by furafylline. Pretreatment of individual human liver microsomes with either 200 mM furafylline
or DMSO control vehicle (1.3% v/v) was conducted in the presence of
an NADPH regenerating system in 100 mM KPO4 at 377C. As described
previously, furafylline is a specific and irreversible inhibitor of CYP1A2
(Kunze and Trager, 1993). The aforementioned concentration of furafylline
in the incubation mixtures (200 mM) has been previously shown to have
no effect on CYP3A4 activity (Gallagher et al., 1994). The microsomal
incubations (approximately 5 mg protein/ml) were allowed to incubate for
20 min, at which time 60-ml aliquots were withdrawn for assay of AFB1
oxidation.
Data analysis. Values reported represent the mean of triplicate incubations. Kinetic parameters for AFB1 oxidation were determined by quantitating the initial rates of formation of AFBO, AFM1 , and AFQ1 in the incubation mixtures. Michaelis parameters and statistics were determined for
CYP1A2-catalyzed AFB1 oxidation in human liver and cDNA-expressed
microsomes by nonlinear regression analysis of the experimental data using
SYSTAT software for the Macintosh computer. CYP 3A4 kinetics were
analyzed by nonlinear regression using the general allosteric model, the
Hill equation (see Results), also using SYSTAT software for the Macintosh
computer.
RESULTS
Aflatoxin B1 Metabolism by cDNA-Expressed Human
CYP1A2
Two metabolic products, AFBO and AFM1 , were formed
in assay incubations using commercially available human
CYP1A2 cDNA-expressed microsomes. These products
were formed in a 1:2.5 (AFM1 :AFBO) ratio (Fig. 1A). Product formation for both products conformed to simple Michaelis–Menten kinetics (Figs. 1A–1C). The apparent Km’s
for AFBO (41 { 4 mM) and AFM1 (36 { 9 mM) calculated
by nonlinear regression (Table 1) were similar as expected
for a ‘‘single enzyme, single substrate’’ system which produces two products. The Vmax values for AFBO and AFM1
formation were 2.63 { 0.06 and 0.92 { 0.06 nmol min01
nmol01, respectively, based on the spectral P450 content
provided by the manufacturer (Gentest Inc.) for the enzyme
lots used.
Aflatoxin B1 Metabolism by cDNA-Expressed Human
CYP3A4
Two AFB1 metabolites, AFQ1 and AFBO, were formed in
CYP3A4 cDNA-expressed microsomal incubation mixtures.
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FIG. 1. The initial rates of AFB1 metabolite formation in lymphoblastoid microsomes stably expressing human CYP1A2 cDNA. (A) V vs S plot
of AFBO and AFM1 formation, (B) Eadie–Hofstee plot of AFBO formation,
(C) Eadie–Hofstee plot of AFM1 formation.
These products were formed in a 10:1 (AFQ1 :AFBO) ratio
(Fig. 2A). A sigmoidal relationship between substrate concentration and product formation rates was observed for both
products (Fig. 2A), in contrast to the hyperbolic Michaelis–
Menten kinetics observed for CYP1A2. Product formation
at high substrate concentrations approached saturation where
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GALLAGHER ET AL.
a 50% increase in substrate concentration from 341 to 512
mM resulted in a 5% increase in product formation (Fig. 2A).
At low substrate concentrations, a more than proportional
increase in reaction rate (1.9-fold) was observed relative to
the increase in substrate concentration (1.5-fold: 20 to 30
mM AFB1 ) suggesting that product formation was greater
than first order with respect to substrate.
One possible interpretation of these results is that AFQ1
and AFBO formation is driven by an allosteric interaction
between substrate AFB1 and CYP3A4. Accordingly, the data
were analyzed by nonlinear regression using the general allosteric model, the Hill equation (Eq. 1). In this equation, n
represents the number of apparent binding sites, K* is composed of the product of affinities for each site and any interaction factors associated with cooperative binding, and Vmax
is the maximum rate when all binding sites are occupied
(Segal, 1975). Due to the large (25-fold) range of product
formation velocities observed it was necessary to employ 1/
Y weighting in order to obtain adequate fit of the data at
the low substrate concentration velocities.
vÅ
Vmaxr(S)n
K* / (S)n
(1)
Eadie–Hofstee plots showing the Hill Equation fit to the
data are shown in Figs. 2B (for AFBO formation) and 2C
(for AFQ1 formation). This plot was used because it readily
exposes the sigmoidal kinetics observed and emphasizes the
second order relationship between substrate and product formation rates at low substrate concentrations. The calculated
Vmax values were 61 nmol AFQ1 formed min01 nmol01 and
6 nmol AFBO formed min01 nmol01 (Table 2). Total satura-
TABLE 1
Kinetic Parameters for CYP1A2-Catalyzed AFBO and AFM1
Formation in CYP1A2 cDNA-Expressed Lymphoblastoid and Human Liver Microsomesa
Microsome
Metabolite
CYP1A2
AFBOa
AFM1*
AFBOa
AFM1*
AFBOa
AFM1*
AFBOa
AFM1*
HLM 105
HLM 109
HLM 125
Km (ASE)b
(mM)
41
36
32
32
47
20
35
28
(4)
(9)
(2)
(3)
(7)
(4)
(1)
(1)
Vmax (ASE) (nmol/
min/nmol P450)
2.63
0.92
0.69
0.16
0.47
0.08
0.36
0.08
(0.06)
(0.06)
(0.02)
(0.01)
(0.02)
(0.01)
(0.01)
(0.01)
Note. * reflects the rate of metabolite formation in uninhibited microsomes. a Reflects the difference of the rates of metabolite formation in
furafylline-inhibited and control (uninhibited) microsomes.
a
Kinetic parameters were calculated by nonlinear regression analysis.
b
Asymptopic standard error.
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FIG. 2. The initial rates of AFB1 metabolite formation in lymphoblastoid microsomes stably expressing human CYP3A4 cDNA. (A) V vs S plot
of AFBO and AFQ1 formation, (B) Eadie–Hofstee plot of AFBO formation,
(C) Eadie–Hofstee plot of AFQ1 formation. B and C include predicted lines
of fit by application of the Hill equation (Eq. 1) to the experimental data.
tion of the enzyme with substrate could not be achieved
because of substrate solubility limitations. Using the same
assumption of optimal activity made earlier for cDNA-expressed CYP1A2 microsomes, the summed products turnover number for AFB1 and CYP3A4 totaled 67 min01. The
parameter n (number of binding sites) in each case was
approximately 1.7 (Table 2), suggesting that two (the next
higher integer value) catalytically important binding events
or sites exist for AFB1 (Segal, 1975). In the event that two
distinct sites are present, the affinity of AFB1 for each site
might be expected to be different. However, a rough approximation of the averaged dissociation constant (K) for the two
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KINETICS OF AFLATOXIN B1 OXIDATION
TABLE 2
Hill Equation Kinetic Constantsa for CYP3A4-Catalyzed AFB1 Oxidation in Human Liver Microsomes
and in CYP3A4 cDNA-Expressed Microsomes
Metabolite
AFQ1
AFBO
Kinetic
constant
CYP3A4
microsomes
HLM 105
HLM 109
HLM 125
Vmax
K* (mM)
n
K (mM)
Vmax
K* (mM)
n
K (mM)
61
3.6
1.7
139
6.2
6.0
1.8
133
16
3.9
1.6
165
1.5
3.6
1.6
189
21
3.5
1.6
146
2.3
3.0
1.6
176
15
5.6
1.7
155
1.4
4.4
1.7
156
(1.6)
(0.6)
(0.1)
(0.2)
(1.2)
(0.1)
(0.5)
(0.7)
(0.1)
(0.1)
(0.5)
(0.1)
(0.5)
(0.5)
(0.1)
(0.1)
(0.4)
(0.1)
(0.7)
(1.9)
(0.1)
(0.1)
(1.3)
(0.1)
a
Vmax expressed as nmol/min/nmol P450, means (ASE) of triplicate determinations; n represents apparent minimum number of substrate binding sites,
means (ASE); K is a composite rate constant that would theoretically equal the substrate concentration that yields half-maximal velocity when N Å 1,
(K Å K*1/n)), expressed in micromolar mean (ASE).
sites can be calculated as 135 mM from the relationship K
Å K*(1/n) (Segal, 1975) (Table 2).
Aflatoxin B1 Metabolism by Human Liver Microsomes
The formation of AFB1 metabolites by human liver microsomes was determined over substrate concentrations ranging
from 0.5 to 512 mM using microsomes isolated from the
livers of three individuals. Mean formation rates from triplicate incubations were determined simultaneously at each
substrate concentration in control microsomes and in the
same microsomes which had been subjected to a brief pretreatment with furafylline to eliminate the CYP1A2 contribution to product formation. In general, the formation of
AFBO and AFM1 was inhibited by furafylline pretreatment,
while AFQ1 formation was resistant to inhibition as noted
in previous experiments (Gallagher et al., 1994). The inhibition of AFBO formation was most marked at low concentrations of substrate (data for HLM105, the preparation with
the highest CYP1A2 content, and HLM109, the preparation
with the highest CYP3A4 content, are presented in Fig. 3).
Consistent with our previous study (Gallagher et al., 1994),
AFM1 formation was inhibited by furafylline at all concentrations of substrate (data not shown).
The formation of AFQ1 in human liver microsomes followed
sigmoidal kinetics (data shown for HLM109 in Figs. 4A–4B)
and was not attenuated by furafylline pretreatment. AFQ1 sigmoidal kinetics were analyzed by nonlinear regression using
the Hill equation, and the calculated parameters Vmax , K*, and
n are reported in Table 2. In each case, application of the Hill
equation provided an excellent fit to AFQ1 formation rates
(Fig. 4B for HLM109). Vmax values expressed relative to total
microsomal P450 content were roughly 25% of the value in
cDNA-expressed CYP3A4 lymphoblastoid microsomes. Based
on CYP3A4 content estimates from Western blot data for
HLM105 and HLM109 (290 and 326 pmol/nmol total P450,
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or 29 and 33%, respectively; K. Thummel, personal communication), the turnover numbers to AFQ1 in these two sets of
liver microsomes were 54 and 65 min01, nearly identical to the
turnover determined for cDNA-expressed CYP3A4, suggesting
that the commercial CYP3A4 cDNA-expressed microsomes
were fully active. Values of K*, K, and n were reasonably
consistent and similar to those in the cDNA-expressed
CYP3A4 microsomes (Table 2). A second order relationship
between substrate concentration and product formation was
observed at low substrate concentrations. For example, mean
AFQ1 formation rates at 8 and 16 mM were 140 and 559 pmol
nmol P45001 min01, respectively, in HLM109, representing a
four-fold increase in rate relative to a two-fold increase in
substrate concentration.
The rates of AFBO formation in control (C, reflecting the
contribution of CYP1A2 and 3A4 in untreated microsomes)
and furafylline-pretreated (F, reflecting primarily CYP3A4)
microsomes were analyzed by Eadie–Hofstee plots. In addition, the difference in mean formation rate (control minus
furafylline-pretreated, C–F, reflecting primarily CYP1A2)
was calculated at each substrate concentration and analyzed
graphically. Fig. 5 shows the Eadie–Hofstee plots for AFBO
formation in HLM109, the microsomal preparation which
contained the highest amount of CYP3A4. Sigmoidal kinetics were observed for AFBO formation in furafylline-pretreated microsomes, suggesting that this residual activity is
principally due to CYP3A4 . The mean rates of AFBO formation at 8 and 16 mM in HLM109 were 17 and 54 pmol
nmol P45001 min01, respectively, representing a 3.2-fold increase in rate relative to a 2-fold increase in substrate
concentration. Hill parameters for AFBO formation in furafylline-pretreated microsomes for HLM109, HLM105, and
HLM125 calculated by nonlinear regression are also presented in Table 2. Vmax values for AFBO, as for AFQ1 formation, were roughly 25% of the values in cDNA-expressed
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GALLAGHER ET AL.
FIG. 3. Effect of furafylline on the inhibition of the initial rates of
AFB1 –8,9-epoxide (AFBO) formation in human liver microsomes. The
assays were conducted in triplicate in the presence or absence of furafylline
as described under Materials and Methods with an initial AFB1 concentration of 0.5–512 mM. The rates of AFBO formation in the presence of
inhibitor were calculated as a percentage of the rates under control conditions. (A) HLM105, and (B) HLM 109.
microsomes, although K* was somewhat larger in the
CYP3A4 preparation (Table 2).
The mean AFBO formation rate difference (C–F) plots produced straight lines indicative of single enzyme Michaelis–
Menten kinetics (data for HLM109 are presented in Fig. 5).
Vmax and Km values for C–F rates in each of the three liver
microsomes were calculated by nonlinear regression and are
given in Table 1. The three Km values fell within a narrow
range (32 to 47 mM) and compared favorably with the Km
observed with the commercial CYP1A2 preparation (41 { 4
mM, Table 1). Vmax values in HLM normalized to total spectral
P450 content ranged from 13 to 26% of the Vmax in the cDNAexpressed CYP1A2 microsomes. Most (but not all) of the formation of this metabolite was abolished in furafylline-pretreated microsomes (data not shown). AFM1 formation in control microsomes also obeyed Michaelis–Menten kinetics. Km
and Vmax values were calculated for AFM1 production and are
presented in Table 1. The Km values for AFM1 formation in
the human liver microsomes (20–32 mM) were similar to the
Km values for AFM1 formation in CYP1A2 cDNA-expressed
microsomes (36 { 9 mM; Table 1).
The furafylline-inhibition data from this and our previous
study (Gallagher et al., 1994) indicate that CYP1A2 and 3A
enzymes contribute to virtually all of the AFBO formation
in human liver microsomes. Furthermore, the excellent fit
of the CYP1A2 data to the Michaelis–Menten equation, and
of the CYP3A4 data to the Hill equation, suggests that these
two models may be employed to predict the in vitro pathway(s) for AFB1 oxidation at substrate concentrations below
assay detection limits. The formation of the toxifying metabolite AFBO in untreated microsomes can be accounted for
as the sum of the CYP1A2 and CYP3A4 contributions using
the following equation composed of a Michaelis–Menten
and Hill term:
V(sum) Å V(1A2) / V(3A4)
CYP3A4 microsomes, and the ratio of AFQ1 to AFBO
formed was approximately 10:1 in each set of microsomes.
Parameters K* and n agreed closely for the human liver
Å
Vmax(1A2) 1 [S] Vmax(3A4) 1 [S]n
/
.
[S] / Km(1A2)
K*(3A4) / [S]n
(2)
FIG. 4. The initial rates of AFQ1 formation in HLM 109. (A) V vs S plot of AFQ1 formation, (B) Eadie–Hofstee plot of AFQ1 formation in HLM109,
with the predicted lines of fit by application of the Hill equation (Eq. 1) to the experimental data.
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0.100 mM substrate concentration, 99% of AFBO formation
is through CYP1A2 (Table 3).
[H3]AFB1-DNA Binding
FIG. 5. Eadie–Hofstee plot of AFBO formation in HLM 109 with the
predicted lines of fit based upon the summed Michaelis–Menten and Hill
equations (Eq. 2). The rates of AFBO formation in uninhibited microsomes
reflect the contribution of all P450’s contributing to AFB1 epoxidation
(primarily CYP1A2 and 3A4, triangles). The rates of AFBO formation
in furafylline-inhibited microsomes primarily reflect the contribution of
CYP3A4 (squares). The rates of CYP1A2-catalyzed AFB1 epoxidation were
calculated by subtracting the rate of AFBO formation in furafylline-inhibited microsomes from the rate of AFBO formation in control microsomes
(circles).
This equation was used to generate the fit to predict the
rate of AFB1 oxidation in HLM at in vivo substrate concentrations (0.100 mM). As observed, the model showed a remarkably high degree of accuracy in predicting the actual
rates of AFB1 oxidation in the measurable range of AFB1
oxidation (data for observed and predicted AFB1 oxidation
at 9.3 and 256 mM AFB1 concentrations in HLM125 are
presented in Table 3). The model predicts that at the target
Two DNA binding experiments were done. In the first
experiment, AFB1 –DNA binding studies were conducted
using high concentrations of human liver microsomes
(HLM105; 0.6 mM P450) as the catalyst for metabolite generation so that detectable AFB1 –DNA adducts could be quantitated at a low substrate concentration (0.133 mM [3H]AFB1 )
reflective of what might occur from dietary exposure in vivo.
To determine the relative contribution of CYP1A2 to DNA
binding at low AFB1 concentrations, parallel incubations
were conducted with control and furafylline-pretreated microsomes. Control and furafylline-pretreated microsomes
catalyzed the binding of 1.71 and 0.085 pmol of AFB1 to
DNA (Fig. 6A), suggesting that CYP1A2 is the dominant
catalyst responsible for ú95% of the formation of AFB1 –
DNA adducts in this HLM preparation at this low substrate
concentration. The second experiment was done using
cDNA-expressed human CYP1A2 and 3A4 microsomes at
nearly identical concentrations of CYP1A2 (0.041 mM) and
CYP3A4 (0.046 mM) and a substrate concentration of 15.3
mM [3H]AFB1 . This experiment was done at a relatively high
substrate concentration to ensure that we could adequately
detect DNA adducts in both samples and could then compare
DNA binding with AFBO production in the measured range.
As observed in Fig. 6B, total binding to DNA catalyzed by
CYP1A2 and CYP3A4 cDNA-expressed microsomes was
8.3 and 2.68 pmol, respectively, a ratio of 3.1:1. Using the
TABLE 3
Predicted a and Observed Rates of CYP1A2 and 3A4-Catalyzed AFB1 Oxidation in HLM125
AFB1
(mM)
Component
AFBO formation
(pmol/min/nmol
P450) predicted
(observed)
AFQ1 formation
(pmol/min/nmol
P450) predicted
(observed)
AFM1 formation
(pmol/min/nmol
P450) predicted
(observed)
Total
3A4
1A2
SUM
% 1A2
3A4
1A2
SUM
% 1A2
3A4
1A2
SUM
% 1A2
947 (918)
315 (316)
1262 (1234)
25.0% (25.6%)
12 (10)
73 (71)
85 (81)
85.9% (87.7%)
0.007
1.02
1.03
99.3%
10,642 (10,221)
õ5a (0)
10,632 (10,221)
õ1% (0%)
121 (96)
õ5a (0)
121 (96)
õ4% (0%)
0.03
0.00
0.03
0%
õ5a (16)
72 (89)
72 (105)
100% (84.8%)
õ5a (1)
20 (18)
20 (19)
100% (94.6%)
0
0.28
0.28
100.0%
11,589 (11,155)
387 (405)
11,976 (11,560)
3.2% (3.5%)
133 (107)
93 (89)
226 (196)
41.2% (45.4%)
0.04
1.30
1.34
97.0%
256
9.30
0.100*
Note. a below detection limit of assay in human liver microsomes (5 pmol/min/nmol P450). *, all values for 0.100 mM AFB1 are predicted based upon
the above models, due to limits of assay sensitivity.
a
CYP3A4 predicted data modeled by application of the Hill equation (Eq 1), CYP1A2 predicted data modeled by Michaelis–Menten kinetics. SUM
reflects the contribution of CYP1A2 and 3A4 as modeled in Eq. (2).
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tion and the kinetic constants for cDNA-expressed CYP3A4
in Table 2, the rate of total CYP3A4-mediated epoxide production at 15.3 mM was determined to be 105 pmol min01
nmol P45001, which according to Ueng et al. (1995), should
all be in the exo conformation. Thus, the ratio of DNA
binding metabolites (exo-AFBO) formed by CYP1A2 and
3A4 at 15.3 mM as determined by HPLC analysis of metabolites is 3.4:1, which is in very close agreement with the
measured ratio (3.1:1) obtained in the DNA binding experiment (Fig. 6B).
DISCUSSION
FIG. 6. Role of CYP1A2 and 3A4 in AFB1 –DNA binding. (A) HLM
105 as the enzyme source. The rates of CYP1A2-catalyzed AFB1 –DNA
binding were calculated by subtracting the rate of AFB1 –DNA binding in
furafylline-inhibited microsomes from the rate in control microsomes. The
reactions were initiated by the addition of [3H]AFB1 (3685 mCi/mmol in
DMSO) dissolved in unlabeled AFB1 to give a final concentration of 0.133
mM AFB1 . (B) Human CYP1A2 and 3A4 cDNA-expressed microsomes as
the enzyme source. The reactions in B were initiated by the addition of
[3H]AFB1 (82.6 mCi/mmol in DMSO) dissolved in unlabeled AFB1 to give
a final concentration of 15.3 mM AFB1 .
Michaelis–Menten equation and the kinetic constants for
cDNA-expressed CYP1A2 in Table 1, the rate of total (exo
plus endo) CYP1A2-mediated AFBO production at 15.3 mM
AFB was determined to be 710 pmol min01 nmol P45001.
According to Ueng et al. (1995), human CYP1A2 produces
an approximately equivalent amount of exo- and endoAFBO, and only exo-AFBO binds to DNA (Iyer et al., 1994).
Thus, correcting for endo formation, the rate of exo-AFBO
production by CYP1A2 in this experiment would be approximately 355 pmol min 01 nmol P45001. Using the Hill equa-
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Previous studies have shown that CYP1A2 and 3A4 are
the most important CYP isoforms responsible for AFB1 metabolism in human liver microsomes (Raney et al., 1992;
Gallagher et al., 1994; Ueng et al., 1995). However, the
relative importance of CYP1A2 and 3A4 with respect to
AFB1 activation remains an area of debate. Deciphering the
relative contribution of these two enzymes toward AFB1
oxidation is complicated by the following observations: (1)
both enzymes produce activation (AFBO) and detoxification
(AFQ1 by CYP3A4, AFM1 by CYP1A2) products, (2) the
two enzymes exhibit different affinities for the substrate
AFB1 , and (3) there is evidence of a nonlinear relationship
for CYP3A4 between substrate concentration and AFBO
and AFQ1 formation (Gallagher et al., 1994). Although the
nonlinear kinetics of CYP3A4-mediated AFB1 oxidation, as
well as the allosteric effects of other compounds such as
5,6-benzoflavone on CYP3A4 have been previously reported
(Ramsdell et al., 1991: Raney et al., 1992), the implications
of these phenonema with respect to human AFB1 metabolism
have not been fully explored. This study examined the kinetics of AFB1 metabolism by constituitively expressed cytochromes P4501A2 and 3A4 in human liver to determine
which of those enzymes are responsible for the formation
of each of the phase 1 metabolites AFBO, AFM1 , and AFQ1
at the relatively low dietary concentrations of AFB1 found
in vivo.
Our approach was to first determine the kinetics of product
formation catalyzed by the individual cDNA-expressed P450
enzymes 3A4 and 1A2. The results of these studies were
then used to examine the kinetics of product formation in
human liver microsomes using the selective irreversible
CYP1A2 inhibitor furafylline to test the hypothesis that
most, if not all, of these products are formed by the actions
of CYP3A4 and 1A2. cDNA-expressed CYP1A2-catalyzed
product formation was found to obey hyperbolic Michaelis–
Menten kinetics and the ratio of AFM1 to AFBO formed
in the incubation mixtures was 1:2.5. In contrast, product
formation to AFQ1 and AFBO catalyzed by cDNA-expressed
CYP3A4 obeyed sigmoidal, rather than hyperbolic, kinetics.
Product formation rates were accurately predicted by the
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KINETICS OF AFLATOXIN B1 OXIDATION
Hill equation in cDNA-expressed CYP3A4 microsomes and
furafylline-pretreated human liver microsomes. Examination
of the Hill equation parameters Vmax , n, and K* suggested
that AFB1 has a modest affinity for CYP 3A4, relative to
CYP1A2. The ratio of AFQ to AFBO formed by CYP3A4
was 10:1 and the summed product turnover numbers (Vmax)
was quite high (67 min01).
The sigmoidal relationship between CYP3A4-catalyzed
product formation rates and AFB1 concentration suggests
that an allosteric interaction takes place between the enzyme
and substrate. Similarly, the formation of temazepam from
diazepam, a CYP3A4-mediated pathway, also follows atypical formation kinetics and is well fitted by a sigmoid Vmax
model equivalent to the Hill equation (Andersson et al.,
1994). This equation assumes that multiple substrate binding
sites exist on the enzyme and applies (in its strictest form)
to the situation where product is formed from the dimeric
complex. Estimates for the number of substrate binding sites,
n, ranged from 1.6 to 1.8. Because estimated Hill equation
parameters often return estimates of n somewhat less than
the next highest integer number of binding sites, it appears
that two catalytically important binding sites for AFB1 exist
within the enzyme. This conclusion is strongly supported by
the heterotropic activation experiments reported by Shou et
al. (1994), the results of which suggest the coincident presence of two different substrates (phenanthrene and 7,8benzoflavone, aNF) in or about the active site of CYP3A4.
One highly important consequence of the sigmoidal kinetics associated with CYP3A4 and AFB1 is that the rate of
product formation falls off precipitously (approximately second order at lower concentrations of substrate) as the substrate concentration is reduced (Eq. 1). This is not true for
CYP1A2, which obeys Michaelis–Menten kinetics (a first
order relationship between substrate concentration and product formation). Equation 2 accurately describes the combined contribution of these two enzymes to AFBO formation
in human liver microsomes down to the lowest concentrations of substrate (2 mM) for which product formation can
be accurately measured in human liver microsomes. A projection of the effect of even lower substrate concentrations
(0.1 mM) on the enzymatic origin of AFBO formed indicates
that substantially all AFBO is formed by CYP1A2 (Table
3). Although the kinetic data suggest that CYP1A2 will be
the predominant P450 involved in total epoxide production
at low substrate concentrations, only the fraction of total
epoxide that represents the exo conformer will bind to DNA
(Iyer et al., 1994). In essence, formation of the endo conformer represents a detoxification pathway in regard to genotoxicity. Ueng et al. (1995) estimated that approximately
50% of CYP1A2-generated AFBO production is in the exo
conformation. Assuming that 50% of CYP1A2-mediated
AFBO production was in the nongenotoxic endo conformation, the total amount of exo conformer produced by
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CYP1A2 still greatly exceeds that of CYP3A4 at substrate
concentrations below 1 mM. Thus, at the low substrate concentrations which probably occur in vivo, the formation of
AFBO, as well as AFB1 clearance, are predicted to be dominated by CYP1A2. DNA binding experiments conducted
at a moderate AFB1 concentration (15.3 mM) with cDNA
expressed CYP1A2 and 3A4 demonstrated that measured
rates of AFB–exo-epoxide production agreed well with
AFB–DNA binding. Even at the relatively high substrate
concentration of 15.3 mM, CYP1A2 formed approximately
three times as much AFB–exo-epoxide and generated three
times as much DNA binding as an equivalent amount of
cDNA-expressed CYP3A4.
The sensitivity of our UV and fluoresence-based HPLC
assay limited our kinetic studies to substrate concentrations greater than 2 mM AFB1 , so it was not possible to
directly examine the rate of product formation by HPLC
at submicromolar AFB concentrations. However, using
radiolabeled AFB, it was possible to determine the relative
contribution of CYP1A2 and 3A4 to AFB – DNA binding
in human liver microsomes at submicromolar concentrations. Indeed, the AFB 1 – DNA binding studies using control and furafylline-inhibited human liver microsomes at
an AFB1 concentration of 0.13 mM found that ú95% of
DNA binding could be attributed to CYP1A2 activation
and are consistent with the kinetic model predictions. This
DNA binding experiment strongly supports the hypothesis
that CYP1A2 is responsible for the vast majority (ú95%)
of AFB1 binding to DNA at low concentrations of substrate likely encountered in the human diet. Although
CYP3A4 is generally expressed at a level two to three
times greater than CYP1A2 (Guengerich and Turvy,
1991), and has higher selectivity for exo-epoxide production, the apparently low affinity of CYP3A4 at the submicromolar hepatic AFB concentrations that would result
from dietary intake make it unlikely that CYP3A4 will
contribute significantly to the in vivo activation of AFB1
in most individuals. Although it is possible that CYP enzymes other than 1A2 and 3A4 can contribute to AFB
activation at very low substrate concentrations in vivo, in
the present study CYP1A2 and 3A4 accounted for the vast
majority of AFB activation in human liver microsomes at
all substrate concentrations examined, and furafylline, a
specific CYP1A2 inhibitor, inhibited nearly all exogenous
AFB – DNA binding from human microsomes at a very
low (0.13 mM) AFB1 concentration.
Because AFB1 is highly lipophilic, it is difficult to know
how nominal concentrations of AFB in in vitro microsomal
preparations relate to concentrations in vivo. However, it
seems highly unlikely that, after lipid partitioning, the concentration of AFB at the CYP enzyme sites would be higher
in vivo than in vitro, because the fraction of nonmicrosomal
lipid in vivo is probably much greater than that in vitro.
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GALLAGHER ET AL.
Thus, unless specific accumulation processes for AFB exist
in vivo that are not functional in microsomal fractions, it
seems unlikely that lipid partitioning of AFB in vivo would
increase the concentration of AFB at the site of enzyme
action, relative to that determined in vitro.
In experiments using purified human CYP1A2 and 3A4
proteins that were expressed in a bacterial expression system,
purified, and reconstituted, Ueng et al. (1995) reported that
CYP3A4 is a major human liver P450 enzyme involved in
AFB1 activation. Those authors also reported that CYP1A2
formed less AFBO than did CYP3A4 and that CYP1A2 is
less effective at producing genotoxic products (exo-AFBO)
than 3A4. Based upon their studies, the authors concluded
that CYP1A2 plays a lesser role in AFB1 activation than
does CYP3A4. The present study and that of Ueng et al.
(1995) are highly consistent with respect to the rates of
CYP3A4-catalyzed AFB1 oxidation and also with respect to
CYP3A4 product ratio formation (AFBO:AFQ1 ). The discrepancies between the two studies may be a result of the
different sources of P450’s used among the two experiments.
The reconstitution system of Ueng et al. (1995) was rather
complex and cannot be considered similar to the in vitro
system employed in the present study. Furthermore, the
source of the human CYP1A2 cDNAs used in the experiments of Ueng et al. (1995) contained a modified N-terminus
(Sandhu et al., 1994) and therefore may have exhibited
somewhat different catalytic activities from the CYP1A2
cDNA-expressed microsomes used in our study.
The results of molecular epidemiological studies of urinary AFB1 biomarkers in middle-aged men in Shanghai,
China exposed naturally via the diet to AFB1 indicate that
the presence of the AFB-N7-guanine adduct is a very useful
marker of AFB1 exposure and the best predictor of hepatocellular carcinoma risk (Groopman, 1994; Groopman et al.,
1988, 1992a,b; Groopman and Donahue, 1988). In a followup study, the relative cancer risk associated with the presence
of AFB1 , AFM1 , AFQ1 , AFG1 , or AFP1 were all statistically
significant when the AFB-N7-guanine adduct was also present (Qian et al., 1994). Comparable results have been obtained for AFB1 exposure biomarkers when analyses were
adjusted for potential confounders such as the presence of
positive hepatitis B antigen status and cigarette smoking
(Qian et al., 1994; Ross et al., 1992). If the in vitro kinetic
models employed in the present study hold true for in vivo
dietary AFB1 exposure, then one would expect a high degree
of correlation between the presence of urinary AFB1 –N7
adducts and AFM1 concentrations since these two metabolites are due to CYP1A2. Accordingly, the relatively high
urinary AFM1 concentrations reported by Qian et al. (1994)
indicate that it is a major AFB1 metabolite in humans exposed to AFB1 in vivo, which is consistent with our in vitro
studies. Furthermore, AFB1 –serum albumin adducts (presumably derived from initial AFBO formation) are highly
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correlated with AFM1 excretion in individuals with high
dietary AFB1 exposure (Gan et al., 1988), further supporting
the hypothesis that CYP1A2 plays a dominant role in AFB1
metabolism in vivo. The ratio of activation:inactivation products catalyzed by CYP1A2 (roughly 2.5:1, AFBO:AFM1 )
and CYP3A4 (1:10; AFBO:AFQ1 ) is likely to be a key
determinant of the pathway and biological consequences of
in vivo AFB1 exposure. However, it may be inappropriate
to consider AFM1 formation by CYP1A2 as solely a detoxification process. Data obtained from mutagenicity studies
have shown that AFM1 can be genotoxic when metabolized
by liver cells in culture (Loveland et al., 1988) due to oxidation of the 8,9-double bond to produce AFM–8,9-epoxide
(Bujons et al., 1995). Thus, if significant AFM1 oxidation
occurs in vivo, then urinary AFM1 output would underestimate CYP1A2-mediated AFB1 metabolism.
If one considers the alternative hypothesis that CYP3A4
is the dominant in vivo catalyst for the formation of AFBO
at relevant dietary concentrations of AFB1 , then it follows
that AFQ1 should be the major metabolite observed in vivo.
Based upon this study and previous work (Raney et al.,
1992a; Gallagher et al., 1994; Ueng et al., 1995), in vivo
AFQ1 formation would be at least 10-fold greater than the
sum of all AFBO-derived metabolites (AFB1 –guanine adducts, AFB1 –serum albumin adducts, etc.), and even higher
than AFM1- and AFM1-derived metabolites. Although none
of the molecular biomarker studies in humans to date has
identified AFQ1 as a predominant metabolite of AFB1 , quantitative data on human AFQ1 excretion are very limited.
Improved analytical methodology for the detection of urinary AFQ1 is needed to properly address this issue. The
origin of urinary AFQ1 is also confounded by the presence
of intestinal enterocytic CYP3A4 which catalyzes intestinal
AFB1 –DNA binding (Kolars et al., 1994). If, as our data
suggest, CYP3A4 preferentially forms an AFB1 detoxification product (AFQ1 ) in vivo, then high levels of CYP3A4
expression may actually confer protection against AFB1 hepatocarcinogenesis. In the absence of CYP1A2, CYP3A4 may
still serve as the primary source of AFBO, even though the
relative rate of detoxification to AFQ1 would be higher.
In conclusion, our in vitro kinetic studies of AFB1 oxidation and DNA binding suggest that the dominant route for
in vivo AFB1 activation at dietary concentrations obtained
in human liver is primarily through CYP1A2. While it appears from these in vitro data that CYP3A4 is unlikely to
be a major source of AFB1 activation in the liver in vivo,
evidence that both CYP1A2 and 3A4 are involved in AFB1
metabolism in vivo is substantiated by biomarker studies
indicating the presence of AFM1 and AFQ1 in the urine of
individuals exposed to dietary AFB1 . Unfortunately, the actual urinary and fecal levels of these two metabolites, (in
particular, AFQ1 and possible secondary metabolites) following exposure to AFB1 are not known. Thus, the relative
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ratio of these two metabolites in individuals exposed to dietary AFB1 , a key ratio, is also unknown. The importance
of CYP3A4 in the metabolism of environmental chemicals
and drugs, coupled with the potential for chemical–chemical
interactions, necessitates further investigations into the kinetics of substrate activation in vivo.
ACKNOWLEDGMENTS
605
guanine in human urine obtained in the Gambia, West Africa. Cancer
Epidemiol. Biomarkers Prev. 1, 221–228.
Groopman, J. D., Jiaqi, Z., Donahue, P. R., Pikul, A., Zhang, L., Chen,
J.-S., and Wogan, G. N. (1992b). Molecular dosimetry of urinary aflatoxin-DNA adducts in people living in Guangxi Autonomous Region,
People’s Republic of China. Cancer Res. 52, 45–52.
Guengerich, F. P., and Turvy, C. G. (1991). Comparisons of levels of several human microsomal cytochrome P450 enzymes and epoxide hydrolase
in normal and disease states using immunochemical analysis of surgical
liver samples. J. Pharmacol. Exp. Ther. 256, 1189–1194.
This research was supported in part by NIH Grants ES-05780, ES07033, GM 47850, and GM 32165. E.P.G. is supported in part by an
NIH Postdoctoral Fellowship in Environmental Pathology and Toxicology (No. T32ES-07032).
Hall, A. J., and Wild, C. P. (1994). Epidemiology of aflatoxin-related disease. In The Toxicology of Aflatoxins: Human Health, Veterinary and
Agricultural Significance (D. L. Eaton and J. D. Groopman, Eds.), pp.
233–258. Academic Press, New York.
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