Cooperative effects for CYP2E1 differ between styrene and its metabolites

Article abstract of DOI:10.3109/00498254.2012.760764

1. Cooperative interactions are frequently observed in the metabolism of drugs and pollutants by cytochrome P450s; nevertheless, the molecular determinants for cooperativity remain elusive. Previously, we demonstrated that steady-state styrene metabolism by CYP2E1 exhibits positive cooperativity. 2. We hypothesized that styrene metabolites have lower affinity than styrene toward CYP2E1 and limited ability to induce cooperative effects during metabolism. To test the hypothesis, we determined the potency and mechanism of inhibition for styrene and its metabolites toward oxidation of 4-nitrophenol using CYP2E1 Supersomes and human liver microsomes. 3. Styrene inhibited the reaction through a mixed cooperative mechanism with high affinity for the catalytic site (67μM) and lower affinity for the cooperative site (1100μM), while increasing substrate turnover at high concentrations. Styrene oxide and 4-vinylphenol possessed similar affinity for CYP2E1. Styrene oxide behaved cooperatively like styrene, but 4-vinylphenol decreased turnover at high concentrations. Styrene glycol was a very poor competitive inhibitor. Among all compounds, there was a positive correlation with binding and hydrophobicity. 4. Taken together, these findings for CYP2E1 further validate contributions of cooperative mechanisms to metabolic processes, demonstrate the role of molecular structure on those mechanisms and underscore the potential for heterotropic cooperative effects between different compounds.

Full text of DOI:10.3109/00498254.2012.760764

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Xenobiotica, 2013; 43(9): 755–764
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RESEARCH ARTICLE
Cooperative effects for CYP2E1 differ between styrene and its
metabolites
Jessica H. Hartman1,2, Gunnar Boysen3, and Grover P. Miller1
2
1Department of Biochemistry and Molecular Biology, University of Arkansas for Medical Sciences, Little Rock, AR, USA, Department of Chemistry,
University of Arkansas at Little Rock, Little Rock, AR, USA, and 3Department of Occupational Health and Safety, University of Arkansas for Medical
Sciences, Little Rock, AR, USA
Abstract
Keywords
1. Cooperative interactions are frequently observed in the metabolism of drugs and pollutants
by cytochrome P450s; nevertheless, the molecular determinants for cooperativity remain
elusive. Previously, we demonstrated that steady-state styrene metabolism by CYP2E1
exhibits positive cooperativity.
Allostery, cooperativity, CYP2E1, inhibition,
P450 2E1, styrene
History
2. We hypothesized that styrene metabolites have lower affinity than styrene toward CYP2E1
and limited ability to induce cooperative effects during metabolism. To test the hypothesis,
we determined the potency and mechanism of inhibition for styrene and its metabolites
toward oxidation of 4-nitrophenol using CYP2E1 SupersomesÕ and human liver
microsomes.
Received 10 November 2012
Revised 12 December 2012
Accepted 17 December 2012
Published online 18 January 2013
3. Styrene inhibited the reaction through a mixed cooperative mechanism with high affinity for
the catalytic site (67 mM) and lower affinity for the cooperative site (1100 mM), while
increasing substrate turnover at high concentrations. Styrene oxide and 4-vinylphenol
possessed similar affinity for CYP2E1. Styrene oxide behaved cooperatively like styrene, but
4-vinylphenol decreased turnover at high concentrations. Styrene glycol was a very poor
competitive inhibitor. Among all compounds, there was a positive correlation with binding
and hydrophobicity.
4. Taken together, these findings for CYP2E1 further validate contributions of cooperative
mechanisms to metabolic processes, demonstrate the role of molecular structure on those
mechanisms and underscore the potential for heterotropic cooperative effects between
different compounds.
Introduction
P450s especially CYP1A2 (Isin et al., 2008; Miller &
ˇ
´
Guengerich, 2001), CYP2C9 (Liu et al., 2005; Remınek &
Glatz, 2010) and CYP3A4 (Kapelyukh et al., 2008; Roberts
et al., 2011; Woods et al., 2011), as extensively reviewed
elsewhere (Atkins, 2006; Denisov et al., 2009; Niwa 2008).
Consequently, identification of the underlying mechanisms
for these processes is necessary for establishing a biochemical
explanation for their biological impacts on drug and pollutant
metabolism.
Oxidation by cytochrome P450 enzymes is the primary
metabolic pathway for many xenobiotic compounds including
drugs and pollutants and thus, identifying suitable models for
interpreting and predicting those metabolic reactions is
critical to advancing pharmacological and toxicological
research (Guengerich, 2007). In many cases, the Michaelis–
Menten mechanism explains reported kinetic profiles and
provides parameters for assessing the metabolic clearance of
compounds. Nevertheless, it has become increasingly clear
that P450 metabolism of compounds can be more complex
involving homo- and heterotropic cooperative (allosteric)
interactions. In fact, these non-Michaelis–Menten mechan-
isms may be more of the rule than the exception for many
Although less characterized than other P450s, CYP2E1 is
similarly capable of metabolizing small drugs and pollutants
through cooperative mechanisms. Aromatic and heterocyclic
compounds inhibit CYP2E1 activity through uncompetitive
and mixed inhibition mechanisms, which require two binding
sites for substrate and inhibitor (Collom et al., 2008;
Hargreaves et al., 1994). The kinetic profiles for several
CYP2E1 substrates demonstrate positive cooperativity such
as styrene, m-xylene and 7-ethoxycoumarin (Hill coefficients
1.7, 1.4 and 1.6, respectively) (Harrelson et al., 2008;
Hartman et al., 2012; Spatzenegger et al., 2003). Similarly,
the kinetic profile for CYP2E1 O-deethylation of phenacetin
deviates from linearity at low substrate concentrations,
Address for correspondence: Grover P. Miller, Department of Biochem-
istry and Molecular Biology, University of Arkansas for Medical
Sciences, 4301 W. Markham, Slot 516. Little Rock, AR 72205, USA.
Tel: 501.526.6486. Fax: 501.686.8169. E-mail: MillerGroverP@
uams.edu

756 J. H. Hartman et al.
Xenobiotica, 2013; 43(9): 755–764
metabolic efficiency. The transition between the catalytic
cycles results in the observed positive cooperativity in the
kinetic profile and occurs within reported styrene concentra-
tions in the blood of workers exposed to this pollutant, and
thus this CYP2E1 mechanism may be relevant in vivo. During
oxidative metabolism of styrene, the molecule becomes more
polar and increases in steric bulk. These changes in structure
conflict with the known preference for CYP2E1 to bind
hydrophobic compounds without steric hindrance (Backes
et al., 1993; Miller, 2008; Porubsky et al., 2008). Therefore,
we hypothesize that oxidative products of styrene metabolism
possess decreased affinity toward CYP2E1 and are unable to
induce cooperative effects during metabolism.
We tested this hypothesis by identifying the inhibitory
potency and mechanism for styrene metabolites using
CYP2E1 Supersomes and human liver microsomes pooled
from 150 donors as a model for liver metabolism (Crespi,
2009). We chose oxidation of 4-nitrophenol as a reporter
reaction for these inhibition studies to avoid interference
between styrene metabolites as inhibitors and those generated
during styrene metabolism. Moreover, 4-nitrophenol binds to
catalytic and cooperative sites and thus its metabolism is
sensitive to inhibitory styrene metabolites binding to both
sites. We initially screened the inhibitory potential of styrene
metabolites through IC50 studies. Those results were used to
design and carry out subsequent steady-state kinetic inhibition
studies with the styrene metabolites. Inhibition kinetic
profiles were fit globally to 10 different possible mechanisms
to identify the most probable one involving binding at
catalytic and/or cooperative sites. The role of hydrophobicity
in driving these interactions was determined through
correlative analysis between the partition coefficient (log P)
of the compounds and the corresponding apparent binding
constants. Taken together, these approaches demonstrate
oxidative metabolites of styrene possess altered affinity and
mechanism of interaction with CYP2E1 and thus, provide
insights toward the significance of molecular structure in
cooperativity during CYP2E1 catalysis of metabolic
reactions.
Figure 1. Metabolic pathways for styrene.
although the authors did not investigate the possibility of
cooperativity (Venkatakrishnan et al., 1998). Unlike those
substrates, the metabolism of 4-nitrophenol is not sigmoidal,
and instead displays substrate inhibition (negative coopera-
tivity) for this commonly used marker substrate for CYP2E1
(Koop, 1986). We were the first to provide a mechanistic
explanation for the 4-nitrophenol kinetic profile through a
cooperative mechanism for CYP2E1. Low concentrations
favor 4-nitrophenol binding to a catalytic CYP2E1 site and
undergoing oxidation to 4-nitrocatechol. At higher concentra-
tions, 4-nitrophenol binds to a second, cooperative effector
site, which leads to a catalytically inactive CYP2E1 complex
(Collom et al., 2008). Similarly, homotypic negative coop-
erative effects for aniline were investigated through molecular
dynamics, whereby binding of a second substrate molecule to
a cooperative effector site led to reorientation of crucial active
site residues resulting in negative cooperativity (Li et al.,
2011). Despite insight into some cooperative interactions, the
molecular determinants that favor Michaelis–Menten or
cooperative metabolism of compounds by CYP2E1 still
remain unknown.
Materials and methods
Materials
An investigation of styrene and its metabolites provides an
excellent opportunity to probe the role of structure in favoring
occupancy of CYP2E1 catalytic and cooperative sites. Styrene
is a common industrial pollutant and potential carcinogen
(Barale, 1991; Bond & Bolt, 1989; National Toxicology
Program, 2008). Styrene is oxidized primarily by CYP2E1 to
the genotoxin styrene oxide and, to a lesser extent, 4-
vinylphenol (Figure 1; Bond & Bolt, 1989). Styrene oxide
then undergoes hydrolysis to the less toxic metabolite 1,2-
phenylethanediol (styrene glycol) through action catalyzed by
epoxide hydrolase. Our studies with CYP2E1 Supersomes and
human liver microsomes demonstrated that steady-state
metabolism of styrene by CYP2E1 exhibits positive coopera-
tivity (Hartman et al., 2012). At low styrene concentrations,
weak styrene binding to CYP2E1 leads to inefficient
oxidation of the pollutant. As the styrene concentration
increases, a second molecule binds to CYP2E1 causing an
increase in affinity for substrate and subsequent increase in
All chemicals used in this study were ACS grade or higher.
The following chemicals were purchased from Sigma-Aldrich
(St. Louis, MO): styrene (substrate), styrene glycol (product),
benzyl alcohol (internal standard), 4-nitrophenol (substrate),
4-nitrocatechol (product), 4-nitroanisole (internal standard)
and most inhibitors (8,9-styrene oxide, (R)-1,2-phenylethane-
diol and (S)-1,2-phenylethanediol. The inhibitors (R/S)-1,2-
phenylethanediol and 4-vinylphenol were purchased from
Fisher Scientific (Wilmington, MA). Methanol (100%) was
purchased from AAPER (Shelbyville, KY). Pooled human
liver microsomes (HLM150s) and human CYP2E1
(SupersomesÕ) were purchased from BD Biosciences (San
Jose, CA). Supersomes are recombinant cDNA-expressed
human cytochrome P450 enzyme prepared from the baculo-
virus-infected insect cell system. These insect cell micro-
somes contain supplemental cDNA-expressed human
reductase and cytochrome b5.

DOI: 10.3109/00498254.2012.760764
CYP2E1 cooperativity toward styrene and its metabolites 757
IC50 studies to determine inhibitory potential of
compounds
55 and 165 mM styrene oxide in 50 mM potassium phosphate
pH 7.4, at 37 ꢀC. Due to low solubility in water, all styrene,
styrene oxide and 4-vinylphenol stocks were prepared in
methanol. As in the inhibition studies, all reactions contained
a final concentration of 1% methanol. After 15 min of
incubation, the reactions were diluted 30-fold in 50 mM
potassium phosphate, pH 7.4. A second reaction was then
carried out with the diluted reaction mixtures containing
22 nM CYP2E1 and 50 mM 4-nitrophenol. Reactions were
initiated upon addition of 1.3 mM NADPH and after 30 min,
quenched with an equal volume of 0.4 N perchloric acid
containing internal standard (25 mM 4-nitroanisole).
Quenched reactions were centrifuged and the resulting
supernatant analyzed by HPLC to quantify formation of the
4-nitrocatechol product. Each experiment was performed in at
least three independent experimental replicates. Under these
experimental conditions, product formation was linear with
respect to time.
We assessed the specific interactions between styrene and its
metabolites and CYP2E1 through IC50 studies using the
oxidation of 4-nitrophenol to 4-nitrocatechol as a marker
reaction for CYP2E1 (Koop, 1986). Either 25 nM CYP2E1
Supersomes or 0.25 mg/mL protein for HLM150s was
incubated with 50 mM 4-nitrophenol and at least seven
concentrations of each inhibitor (plus a negative control
with solvent only) in 50 mM potassium phosphate pH 7.4, at
37 ꢀC. Because of solubility, all inhibitor stocks were
prepared in methanol. All reactions contained a final
concentration of 1% methanol. Reactions were initiated
upon addition of 1 mM NADPH and after 30 min, quenched
with an equal volume of 0.4 N perchloric acid containing the
internal standard (25 mM 4-nitroanisole). Quenched reactions
were centrifuged and the resulting supernatant analyzed by
HPLC to quantify formation of the 4-nitrocatechol product
formed. Each experiment was performed in at least three
independent experimental replicates. Under these experimen-
tal conditions, product formation was linear with respect
to time.
Inhibition kinetics for styrene and its metabolites
toward CYP2E1 activity
We determined the inhibition mechanisms for styrene and its
metabolites toward CYP2E1 to elucidate their mode of
interaction with the enzyme and subsequent effect on activity.
For these reactions, either 25 nM CYP2E1 Supersomes or
0.25 mg/mL protein for HLM150s was incubated with 25, 50,
100, 500, 1000 and 2000 mM 4-nitrophenol in 50 mM
potassium phosphate pH 7.4, at 37 ꢀC. Each set of reactions
for a particular inhibitor was carried out in the presence of at
least three different inhibitor concentrations, along with a set
without inhibitor as a negative control. Reactions were
performed, quenched, and analyzed by HPLC, as described
for the IC50 studies. Each experiment was performed in at
least four independent experimental replicates.
HPLC analysis of 4-nitrophenol oxidation by CYP2E1
We improved a previously published in-house HPLC method
to analyze 4-nitrophenol reactions (Collom, 2007). For this
study, we employed a different column to maintain resolution
of compounds, while eliminating the use of acetonitrile and
decreasing run times. Samples were injected onto a Waters
Breeze HPLC system and resolved using a 4.6 ꢁ 150 mm
Zorbax Eclipse 5 mm XDB-C18 column (Agilent, Santa Clara,
CA) heated to 45 ꢀC under isocratic conditions (40:60 0.1%
acetic acid/H2O:methanol) at a flow rate of 1.2 mL/min. The
elution of 4-nitrocatechol (product), 4-nitrophenol (substrate)
and 4-nitroanisole (internal standard) were monitored at
360 nm. Final 4-nitrophenol concentrations were calculated
from the ratio of product area to internal standard area in the
sample and compared to calibration curves as determined
with authentic standards.
The resulting kinetic profiles for styrene and its metabo-
lites were analyzed globally to identify the most probable
inhibition mechanism toward 4-nitrophenol oxidation by
CYP2E1 using DynaFit software version 3.28 (Biokin Ltd,
Watertown, MA) (Kuzmic, 1996). In order to limit the
number of variable parameters during the analysis of the
inhibition data, the uninhibited steady-state mechanism and
parameters were first obtained. Each inhibition data set
included the uninhibited reaction as a negative control and
thus, we compiled and analyzed all of those results to
determine the kinetic parameters (Vmax, Ks and Kss) for the
uninhibited 4-nitrophenol reaction. The DynaFit script file
used for analysis of the uninhibited data is included in
‘‘Supplementary material’’, with the model discriminations
shown in Table S2a and b. As described previously for the
reconstituted rabbit CYP2E1 system (Collom et al., 2008), a
substrate inhibition mechanism best described the metabolism
of 4-nitrophenol by CYP2E1 Supersomes and HLM150. For
each enzyme system, a plot of the uninhibited kinetic profile
as well as a table of parameters for the uninhibited reactions is
also included in ‘‘Supplementary material’’ (Figure S1 and
Table S1). These parameters were held constant and the
inhibitory parameters (Ki, Ksi and kcat2) treated as variables.
For analysis of the inhibition experiments, data were fit to
one or two-site inhibition mechanisms for 4-nitrophenol
Analysis of IC50 data
For each set of inhibition reactions, the corresponding rates of
product formation were normalized relative to the control rate
in the absence of inhibitor and presence of 1% methanol.
These data were plotted as a function of the log of the
inhibitor concentration and fit to the standard IC50 equation
using GraphPad Prism 4.0 (San Diego, CA). The correspond-
ing IC50 value was used to select metabolite concentrations
for subsequent inhibition kinetic studies.
Assessing CYP2E1 inactivation by styrene oxide
Styrene oxide may reversibly interact with CYP2E1 binding
sites and/or react with the enzyme to alter activity.
Consequently, we assessed the significance of styrene
oxide-mediated inactivation of CYP2E1 by measuring
residual activity following pre-incubation of the enzyme
with the metabolite. For these studies, 667 nM CYP2E1
Supersomes was incubated with 1.3 mM NADPH and 0, 18.3,

758 J. H. Hartman et al.
Xenobiotica, 2013; 43(9): 755–764
metabolism, which involves multiple possible inhibitory
complexes (Figure 2). Although the models varied in
complexity (and number of corresponding parameters),
simpler models were favored over more complex mechanisms
in the Dynafit statistical analysis. The proposed inhibition
mechanisms are based on traditional Michaelis–Menten
mechanisms (competitive, uncompetitive, noncompetitive
and mixed), but necessarily included formation of an inactive
ESS complex due to known substrate inhibition during the
uninhibited steady-state reaction. Consideration of a second
substrate-binding site also opened the door to formation of
ESI complexes, which may be catalytically active. In addition
to those possibilities, other mechanisms included the potential
for cooperative effects on binding between substrate and
inhibitors. Contributions of the EII and EIS complexes
(shown in gray in Figure 2) were not measurable using the
catalytic marker assay. Therefore, Ksi,ap and Ki,ap are
designated as apparent parameters that may include contribu-
tions from multiple possible complexes. In total, there were
ten possible mechanisms involving inhibition at one or two
sites and the possibility of cooperativity from the substrate on
inhibitor binding (Table 1). The DynaFit script file used for
the inhibition model discrimination analyses is included in the
‘‘Supplementary material’’.
Assessment of hydrophobicity in binding to the
CYP2E1 catalytic site
We analyzed the importance of hydrophobicity in mediating
binding interactions between CYP2E1 and the styrene
derivatives as well as 4-nitrophenol. We used parameters
obtained from the preferred model describing binding of
either substrate or inhibitor to the enzyme catalytic site. We
employed Marvin software, ChemAxon (Budapest, Hungary)
to determine the log of the standard octanol-water partition
coefficient (log P) for all compounds, as a measure of their
respective hydrophobicity. We plotted the resulting log P
values against the corresponding log of either the inhibition
constants (Ki) or substrate binding constants (Ks) from the
catalytic studies using GraphPad Prism (San Diego, CA).
Data were fit to a simple linear regression.
Results
IC50 values for styrene and its metabolites reveal
inhibitory potential
We screened the inhibitory potential of styrene and its
metabolites through IC50 studies using 4-nitrophenol as the
marker reaction (Table 2 and Figure 3). For CYP2E1
Supersomes and HLM150, styrene, 4-vinylphenol and styrene
oxide had similar and potent inhibitory properties. Styrene
glycol was a very poor inhibitor, approximately 300-fold
above styrene and the other two metabolites. There was
almost no residual activity remaining at high concentrations
of these compounds with CYP2E1 Supersomes. With
HLM150, somewhat greater residual activity remained at
high concentrations.
Styrene oxide inhibition of CYP2E1 is reversible
We tested the reversibility of styrene oxide inhibition of
recombinant CYP2E1 activity by using 4-nitrophenol oxida-
tion as a reporter reaction following incubation with the
epoxide metabolite. Even at levels three-fold higher than the
measured IC50 for styrene oxide, the enzyme retained all of its
initial activity (Figure 4). The negative control (without any
styrene oxide present) had slightly less activity than the
incubations with styrene oxide, suggesting a slight increase in
the stability of CYP2E1 when a ligand was available to
occupy the catalytic site as reported for other compounds
(Roberts et al., 1995; Song et al., 1989).
Styrene and its metabolites interact with two
CYP2E1 sites during 4-nitrophenol metabolism by
recombinant enzyme.
Figure 2. Possible inhibition mechanisms for CYP2E1 4-nitrophenol
activity by styrene and metabolites. Uninhibited reaction shown in bold;
possible but undetectable complexes shown in gray. E ¼ CYP2E1;
S ¼ 4-nitrophenol; P ¼ product (4-nitrocatechol) and I ¼ styrene and
metabolites.
Through kinetic studies with CYP2E1 Supersomes and
HLM150s, we determined the mechanism of CYP2E1
Table 1. Possible models for inhibition of CYP2E1.
Model
Complexes formed
Cooperative effects
Possible active ESI complexa
Parameters and consequencesa
Competitive
Uncompetitive
Mixed
ES, ESS, EI
ES, ESS, ESI
ES, ESS, EI, ESI
ES, ESS, EI, ESI
No
No
Yes
No
No
Yes
Yes
Yes
Ks, Kss, Ki, kcat
Ks, Kss, Ksi, kcat1, kcat2
Ks, Kss, Ki, Ksi, kcat1, kcat2
Ks, Kss, Ki ¼ Ksi, kcat1, kcat2
?
?
?
Noncompetitive
aIn all models except competitive, ESI may be active or not, hence kcat2. Both possibilities were included in model discrimination studies.

DOI: 10.3109/00498254.2012.760764
CYP2E1 cooperativity toward styrene and its metabolites 759
Table 2. IC50 values for styrene and its metabolites.a
Recombinant CYP2E1
HLM150
Metabolite
IC50 (mM)
Residual activity (%)
IC50 (mM)
Residual activity (%)
Styrene
75 (54–95)
55 (35–75)
24 (15–24)
11 (5.2–16)
13 (5.1–20)
16 (8.7–23)
9.7 (0.0–21)
5.6 (0.0–17)
9.2 (0.0–24)
45 (33–58)
150 (24–280)
38 (26–50)
12 000 (7500–16 000)
15 000 (10 000–20 000)
14 000 (6600–21 000)
20 (16–24)
28 (12–44)
19 (12–26)
26 (19–32)
11 (2.7–18)
18 (6.5–29)
Styrene oxide
4-Vinylphenol
R-Styrene glycol
S-Styrene glycol
R/S-Styrene glycol
11 000 (6200–15 000)
15 000 (9200–22 000)
13 000 (2800–23 000)
aThe nonsymmetrical 95% confidence intervals for parameters are shown in parentheses.
Figure 4. Residual CYP2E1 activity after preincubation with styrene
oxide. For reactions, 670 nM CYP2E1 and 1.3 mM NADPH were
incubated with 0, 18.3, 55 and 165 mM styrene oxide at 37 ꢀC and pH 7.4
for 15 min. Following a 30-fold dilution, the residual activity was
assayed at a final concentration of 22 nM CYP2E1 with 50 mM 4-
nitrophenol and 1.3 mM NADPH. The reported values represent the
average of three experimental replicates, including the mean ꢂ standard
deviation. Further reaction conditions are described in Experimental
Procedures.
inactive ESS complex, so ESI complexes may be inactive or
active. If active, the ESI complex may turn over product at the
same or differing rates from the uninhibited reaction. A
statistical analysis was performed to determine the most
probable mechanism according to the AIC (Akaike
Information Criteron). The resulting statistical outputs are
included in Tables S3(a)–(d) and S4(a)–(d).
Figure 3. IC50 plots for styrene metabolites with CYP2E1 Supersomes
(Panel A) and HLM150 (Panel B). Nonlinear plots represent the IC50
nonlinear regression for the normalized activity versus log of inhibitor
concentration in micromolar. Individual tracings represent different
metabolites. Reactions were performed at least four times at 37 ꢀC and
pH 7.4. Further reaction conditions are described in Experimental
Procedures.
In the studies with CYP2E1 Supersomes (Table 3), the
inhibitory potency was strongest for styrene and 4-vinylphe-
nol, slightly weaker for styrene oxide, and very weak for
styrene glycol, although the compounds interacted with
CYP2E1 through differing mechanisms (Figure 5). Styrene
inhibition of the reaction was best explained through two
mixed cooperative mechanisms (Table S3a) that were
essentially equally probable. In these mechanisms, inhibitor
can bind to either free enzyme or the enzyme-substrate
complex (Figure 2). Similar to the inhibitory mechanism for
4-methylpyrazole (Hartman et al., 2012), substrate was a
cooperative effector on inhibitor binding. However, unlike
inhibition by styrene and its metabolites toward oxidation of
4-nitrophenol. As a foundation for these studies, data for the
uninhibited reaction were plotted for CYP2E1 Supersomes
and HLM150 and the corresponding parameters determined
(Figure S1 and Table S1). The inhibition data for each
compound were fit to ten possible mechanisms for inhibition
of the oxidation of 4-nitrophenol, generalized in Figure 2 and
detailed in Table 1. In these mechanisms, binding of inhibitor
can either lead to formation of an EI complex, an ESI
complex, or both. If both complexes form, heterotropic
cooperative interactions may occur in the form of bound
substrate affecting binding of the inhibitor molecule.
Alternatively, cooperative effects on turnover can be
observed; the substrate 4-nitrophenol is known to form an
that interaction, bound 4-nitrophenol had
a negative
cooperative effect on styrene binding, as reflected by a 10-
fold higher Ksi,ap relative to Ki,ap. In both preferred
mechanisms, the ESI complex was active; in one case, the
kcat of turnover from the ESI complex was greater than that
of the uninhibited reaction (kcat2 > kcat1). In the other case,
the two reaction rates were equal. However, in the first case,

760 J. H. Hartman et al.
Table 3. Apparent inhibition parameters for styrene metabolites toward recombinant CYP2E1 activity.a,b
Xenobiotica, 2013; 43(9): 755–764
c
Inhibitor
LogP
Mechanism
kcat2
Ki,ap (mM)
Ksi,ap (mM)
Styrene
2.71
1.74
2.41
0.58
Mixed cooperative substrate
Mixed cooperative substrate
Mixed cooperative substrate
Competitive
19 (17–21)
19 (17–21)
67 (56–80)
110 (76–170)
47 (33–72)
1100 (700–2000)
1000 (450–10 000)
230 (120–1500)
Styrene oxide
4-Vinylphenol
R/S-Styrene glycol
–
–
29 000 (19 000–52 000)
–
aFigure 2 depicts reaction mechanism used to determine kinetic parameters. Nonsymmetrical 95% confidence intervals for parameters are shown in
parentheses.
bParameters for uninhibited 4-nitrophenol oxidation by recombinant CYP2E1 were Ks 290 mM, Kss 1700 mM and kcat1 19 nmol/min/nmol CYP2E1.
cUnits are nmol/min/nmol CYP2E1.
complex, but the parameters had open confidence intervals.
The other mechanism had an identical kcat for each, and
closed nonsymmetrical intervals at 95% confidence, as shown
in Table 3.
Like styrene and styrene oxide, the preferred inhibition
mechanism for 4-vinylphenol involved binding at the catalytic
and cooperative sites in a mixed inhibition model in which
substrate acted cooperatively (Table S3d). As with the other
compounds, 4-nitrophenol had a negative cooperative effect
on inhibitor binding, such that Ksi,ap > Ki,ap. However, unlike
those compounds, the preferred mechanism for 4-vinylphenol
inhibition involved an ESI complex that was not catalytically
active. The favored mechanism for styrene glycol inhibition
was its role as a simple competitive inhibitor only for the
catalytic site (Table S3c).
Styrene and its metabolites interact with two CYP2E1
sites during 4-nitrophenol metabolism by human liver
microsomes
For human liver microsomes, the observed substrate inhibi-
tion is less pronounced due to contribution from other P450s
at high 4-nitrophenol concentrations (Figure 6). Because of
Figure 5. CYP2E1 Supersomes inhibition kinetic profiles for styrene
this, there was ambiguity in the discriminations between
and its metabolites toward oxidation of 4-nitrophenol. The dashed line
mechanisms and interactions at the cooperative site were
difficult to observe. However, the preferred inhibition
mechanism for styrene was the mixed cooperative substrate
model, in which substrate acted cooperatively on the reaction
as observed for the studies with recombinant enzyme (Table
S4a). The model that had the greatest statistical support was
that with ESI active and kcat1 ¼ kcat2. However, the corre-
sponding parameters for this model had open confidence
intervals. The model with the next highest probability was the
same model but with the kcat1 ¼ kcat2, and closed confidence
intervals. The parameters associated with this model are
shown in Table 4.
and open circles represent the uninhibited reaction; increasing inhibitor
concentration is indicated by darker lines and markers. Specific
inhibitors include (A) 0, 3.7, 11, 33, 99, 300 and 890 mM styrene;
(B) 0, 18, 55, 170 and 500 mM styrene oxide; (C) 0, 8.7, 26 and 78 mM
4-vinylphenol or (D) 0, 4000, 12 000 and 36 000 mM styrene glycol. For
reactions, 25 nM CYP2E1, varying 4-nitrophenol concentrations, and
1 mM NADPH were incubated at 37 ꢀC and pH 7.4 (Experimental
Procedures). The reported values reflect the average of four experimental
replicates, including the mean ꢂ standard deviation. Data were fit to the
most probable mechanism listed in Table 3, which were identified among
10 possibilities (Table 1) using DynaFit software version 3.28 (Biokin
Ltd) (Kuzmic, 1996).
the confidence intervals were open and thus, we could not
properly fit the parameters. Thus, there is essentially one
preferred mechanism for styrene inhibition of 4-nitrophenol
oxidation by CYP2E1.
The discriminations for styrene oxide, styrene glycol, and
4-vinylphenol (Tables S4b, d and c, respectively) showed a
preference for competitive models and mixed cooperative
substrate models; however, the mixed cooperative models had
kinetic parameters with open confidence intervals. The
parameters associated with a competitive inhibition mechan-
ism are reported in Table 4.
Inhibition induced by styrene oxide was best explained
through a mechanism similar to that observed for styrene.
There was an equal probability of two different mixed
inhibition mechanism in which 4-nitrophenol (substrate) acted
as a negative cooperative effector on styrene oxide binding
(Ksi,ap >> Ki,ap); however, they differed on the rates of
catalysis (Table S3b). One mechanism had different product
formation rates for the uninhibited reaction and the ESI
Taken together, the corresponding inhibition constants for
the catalytic site (Ki) from CYP2E1 Supersomes and
HLM150 reactions in Tables 3 and 4 were similar to the
IC50 values in Table 2.

DOI: 10.3109/00498254.2012.760764
CYP2E1 cooperativity toward styrene and its metabolites 761
Hydrophobicity plays dominant role in binding inter-
actions to CYP2E1 catalytic site
hydrophobicity is likely important for protein binding rather
than steric hindrance or other factors.
For CYP2E1 Supersomes and human liver microsomes, the
apparent Kd,ap (Ki,ap for styrene and metabolites and Ks,ap for
4-nitrophenol) were significantly correlated with the log of
the partition coefficient (log P). The correlation had a
coefficient of determination (r2) of 0.85 for CYP2E1
Supersomes and 0.93 for human liver microsomes
(Figure 7). The same trends were observed for each system
with approximately the same magnitude for each. There were
no apparent outliers from this analysis. These correlations
suggest that for the compounds included in this study
Discussion
Herein, we demonstrated the impact of styrene metabolism on
the ability of the molecule to modulate CYP2E1 activity
through a cooperative mechanism. Specifically, we investi-
gated the mode of interaction between CYP2E1 and an array
of molecules, i.e. styrene, its oxidized metabolites (styrene
oxide and 4-vinylphenol), and a secondary metabolite, styrene
glycol. Initial IC50 studies using 4-nitrophenol as a reporter
provided valuable insights on the inhibitory potential of the
compounds. Nevertheless, the simplicity of those experiments
obviated the ability to truly investigate the mechanism of
interaction as revealed by kinetic inhibition studies. Styrene
and its metabolites preferably interacted with CYP2E1
Figure 6. Inhibition kinetic profiles for styrene and its metabolites
toward oxidation of 4-nitrophenol with human liver microsomes
(HLM150). The dashed line and open circles represent the uninhibited
reaction; increasing inhibitor concentration is indicated by darker lines
and markers. Specific inhibitors include (A) 0, 3.7, 11, 33, 99 and
300 mM styrene; (B) 0, 18, 55, 170 and 500 mM styrene oxide; (C) 0, 8.7,
26 and 78 mM 4-vinylphenol or (D) 0, 4000, 12 000, and 36 000 mM
styrene glycol. For reactions, 0.25 mg/mL HLM150, varying 4-
nitrophenol concentrations, and 1 mM NADPH were incubated at
37 ꢀC and pH 7.4 in the presence of inhibitor (Experimental
Procedures). The reported values reflect the average of four experimental
replicates, including the mean ꢂ standard deviation. Data were fit to the
most probable mechanism listed in Table 4, which were identified among
10 possibilities (Table 1) using DynaFit software version 3.28 (Biokin
Ltd) (Kuzmic, 1996).
Figure 7. Importance of hydrophobicity in binding interactions with the
CYP2E1 catalytic site. logP values were calculated using the Marvin
software, ChemAxon (Table 3) were plotted against Kd representing the
inhibition constant Ki for styrene and metabolites or equilibrium constant
Ks for 4-nitrophenol. Data were then fit to a linear regression.
Table 4. Apparent inhibition parameters for styrene metabolites toward CYP2E1 activity with HLM150.a,b
c
Inhibitor
Mechanism
kcat2
Ki,ap (mM)
Ksi,ap (mM)
Styrene
Mixed cooperative substrate
Competitive (mixed cooperative substrate)d
Competitive (mixed cooperative substrate)
Competitive
1.7 (1.5–1.9)
18 (7.6–42)
160 (140–190)
61 (47–83)
58 (21–190)
Styrene oxide
4-Vinylphenol
R/S-Styrene glycol
–
–
–
–
–
–
17 000 (15 000–19 000)
aFigure 2 depicts reaction mechanism used to determine kinetic parameters. Nonsymmetrical 95% confidence intervals for parameters are shown in
parentheses.
bParameters for uninhibited 4-nitrophenol oxidation by HLM150 were Ks ¼ 150 mM, Kss ¼ 11 000 mM and kcat1 ¼ 1.7 nmol/min/mg protein.
cUnits are nmol/min/mg protein.
dProbability for both models was statistically equivalent, but the parameters for the model in parentheses had open confidence intervals.

762 J. H. Hartman et al.
Xenobiotica, 2013; 43(9): 755–764
through cooperative mechanisms dependent on the respective
structure of the molecules. The strongest evidence came from
studies employing CYP2E1 Supersomes, which were devoid
of competing P450s and other possible reactions. Human liver
microsomes recapitulated the findings, although it was not
always possible to define confidence intervals for parameters.
There was some unavoidable ambiguity in the models, which
did not allow true equilibrium constants to be determined;
however, the mechanisms reported herein provide crucial
evidence for multiple CYP2E1 binding sites for styrene and
its metabolites. Collectively, these CYP2E1 studies expanded
the array of known compounds to interact with its cooperative
site and revealed the importance of structure on determining
the impacts on metabolism (Harrelson et al., 2008; Hartman
et al., 2012; Spatzenegger et al., 2003).
to inactivate CYP2E1. Consequently, CYP2E1 remained
active toward 4-nitrophenol and metabolized it through a
new catalytic cycle involving a mixed complex with both
4-nitrophenol and styrene bound to the enzyme.
Oxidation of styrene significantly altered its inhibition
mechanism toward the 4-nitrophenol reaction and not its
affinity toward CYP2E1 sites. Styrene epoxide and
4-vinylphenol inhibited 4-nitrophenol metabolism through a
mixed cooperative mechanism. Similar affinities of these
molecules for the catalytic site to those observed for styrene
(Table 3) indicated epoxidation of the vinyl group or
hydroxylation of the phenyl ring did not alter the ability to
form effective binding contacts. Both alterations in the
structure introduce minimal steric bulk, although they do
introduce some polarity, especially the epoxide moiety. When
4-nitrophenol bound to the catalytic site, there were much less
productive binding interactions with these metabolites at the
cooperative site as suggested for styrene. Nevertheless, the
specific binding modes for styrene oxide and 4-vinylphenol
were different given their impacts on catalysis. Like styrene,
styrene oxide changed the metabolic pathway for 4-nitrophenol
by creating a mixed complex that was catalytically active
(Figure 8). The analogous mixed complex for 4-vinylphenol
was catalytically inactive. In other words, 4-vinylphenol
binding at the cooperative site impacted catalysis more like
4-nitrophenol rather than styrene. It is possible that the
introduction of the hydroxyl group to styrene may create new
binding interactions that are not possible for styrene or its
epoxide. For example, 4-vinylphenol and 4-nitrophenol are
both hydrogen bond donors and acceptors. In following,
phenolic compounds may favor the formation of higher order
but catalytically inactive CYP2E1 complexes, although further
studies are necessary to better establish this relationship.
An alternate but not exclusive inhibition mechanism for
styrene oxide would involve adduct formation between the
metabolite and CYP2E1. Epoxides are electrophilic and are
prone to attack by nucleophilic sites on proteins. We have
shown that treatment of CYP2E1 with 1,2-epoxy-butadiene
resulted in the modification of four residues (Boysen et al.,
2007). Although no adducts between styrene oxide and
CYP2E1 have been reported, exposure to styrene leads to
formation of styrene oxide adducts to albumin and hemoglobin
(Christakopoulos, 1993; Rappaport et al., 1996). If an adduct
formed between styrene oxide and CYP2E1, the modification
of its structure could have blocked 4-nitrophenol access to the
catalytic site and/or compromised interactions with cyto-
chrome P450 reductase, which are necessary for activating
oxygen during catalysis (Guengerich, 2002). Pre-incubation of
styrene oxide with CYP2E1 did not alter activity (Figure 4),
indicating that there are no functional consequences even if
adducts formed between styrene oxide and CYP2E1. In the
absence of covalent modifications, styrene oxide inhibition of
CYP2E1 activity is solely a reversible process.
Styrene inhibition of the oxidation of 4-nitrophenol shared
many mechanistic similarities with our findings for styrene
metabolism, in particular, the association with catalytic and
cooperative effector sites (Hartman et al., 2012). In both
cases, styrene bound to CYP2E1 through two sites – one high
affinity site and another low affinity one. Styrene metabolism
involved an initial weak binding event between styrene and
CYP2E1 followed by
a much stronger second one.
Presumably, the first binding event created a more hydro-
phobic environment in the catalytic site to favor binding of a
second styrene molecule. In the current studies, both of these
binding events for styrene collectively blocked 4-nitrophenol
binding to the catalytic site to inhibit its metabolism.
Consequently, the apparent inhibition constant for styrene
(Ki,ap 67 mM for CYP2E1 Supersomes, Table 3) is comparable
to the high affinity apparent binding constant observed during
styrene metabolism (Ks 110 mM for CYP2E1 Supersomes)
(Hartman et al., 2012). By contrast, when 4-nitrophenol
bound to the catalytic site, the association of styrene with the
cooperative site was rather poor (Ksi,ap 1100 mM for CYP2E1
Supersomes, Table 3). The lack of productive binding
contacts to styrene may be due to a decrease in steric space
and an increase in polarity in the catalytic site because of the
presence of 4-nitrophenol. These findings indicate that the
properties of the compound bound at the catalytic site can
influence binding to the cooperative site.
Similarly, occupancy of the cooperative effector site
impacted substrate binding and catalysis although specific
effects depended on the structure of the respective compounds.
Styrene bound to the cooperative effector site improved the
binding of another styrene molecule at the catalytic site
(Hartman et al. 2012). The formation of favorable hydrophobic
and/or pi stacking interactions may account for the increased
affinity between styrene and CYP2E1. A consequence of this
mode of binding for styrene created a new more efficient
catalytic cycle for CYP2E1, even though styrene did not act
cooperatively on kcat for the reaction (Hartman et al., 2012).
Likewise, styrene bound at the cooperative site produced no
cooperativity on kcat for 4-nitrophenol oxidation; rather, the
effects manifested in changes in the reaction pathway for 4-
nitrophenol and an overall increase in catalytic efficiency as
shown in Figure 8. In the absence of styrene, 4-nitrophenol
bound to the cooperative site to create an inactive complex and
subsequent substrate inhibition. When styrene occupied the
cooperative site, 4-nitrophenol could not associate with the site
Cleavage of styrene oxide significantly altered the
inhibition mechanism and affinity of the compound toward
CYP2E1. Styrene glycol binding was extremely poor and
behaved as competitive binder interacting solely though the
catalytic site. However, this observation does not rule out
possible binding to the cooperative site. Styrene and its
oxidized metabolites bound more poorly to the cooperative

DOI: 10.3109/00498254.2012.760764
CYP2E1 cooperativity toward styrene and its metabolites 763
Figure 8. Stylized depiction of the cooperative mechanism for inhibition of CYP2E1 metabolic reactions. In the case of 4-nitrophenol, substrate binds
to the CYP2E1 catalytic site indicated by the red heme-iron group and forms an active binary complex, which releases product and regenerates the free
enzyme. At higher 4-nitrophenol concentrations, a second substrate molecule binds the enzyme-substrate complex at a distal cooperative site and forms
a dead-end complex. When a cooperative inhibitor such as styrene is also present, it can bind either the free enzyme or the enzyme-substrate complex to
form a binary or (active) ternary complex. This illustrates some of the different types of cooperativity that can occur when a mixture of compounds is
present in the system.
site than the catalytic site. For the glycol metabolite, the
weakness of the initial binding event may preclude observa-
tion of binding at the cooperative site. The opening of the
epoxide ring to form the glycol introduced more steric bulk
and more importantly, increased the polarity of the molecule
leading to the significant decrease in CYP2E1 binding.
The studies performed with human liver microsomes
allowed us to interrogate the potential inhibition mechanisms
in a system that better approximated in vivo conditions. While
4-nitrophenol is more effectively metabolized by CYP2E1,
other P450s can contribute at higher concentrations. Those
data points also played an important role in discriminating
between possible inhibition mechanisms, and thus contribu-
tion from multiple enzymes to observed rates likely account
for difficulties identifying a single preferred inhibition
mechanism. Nevertheless, the preferred mechanisms with
human liver microsomes did recapitulate the findings with
CYP2E1 Supersomes: styrene and its metabolites likely
interact at two sites on CYP2E1 and modulate its activity.
of the molecules. Specifically, these inhibition studies
demonstrated that the occupancy of the cooperative site by
an array of molecules led to alternate modes of interactions
between mixtures of molecules and CYP2E1 than simple
competition for a single catalytic site. For these compounds,
the driving force for binding events including at the
cooperative site was formation of favorable hydrophobic
interactions and not steric clashes. The corresponding
evidence was clearer for binding at the catalytic site than
the cooperative site indicating further studies are necessary to
better characterize its specificity. Once occupied, the
cooperative site impacted CYP2E1 metabolism of molecules
overall through two significant, and not mutually exclusive,
ways. Firstly, occupancy of the cooperative site modulated
binding and the rate of catalysis, and thus steps along the
metabolic pathways to product. Secondly, the binding of
molecules to cooperative site created new, mixed complexes
that altered the trajectory of metabolic pathways and impacted
the overall efficiency of metabolism. There were ambiguities
with proposed models due to the inability to define
parameters, such as inhibition constants at either the catalytic
or cooperative sites rather than treatment as a single constant.
Other biophysical methods including binding studies would
provide a powerful complement to these efforts. Nevertheless,
these findings for CYP2E1 further validate the contributions
Concluding remarks
Styrene and its metabolites interacted with CYP2E1
through cooperative mechanisms; however, the consequences
of those interactions depended on differences in the structure

764 J. H. Hartman et al.
Xenobiotica, 2013; 43(9): 755–764
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Declaration of interest
The authors report no declarations of interest.
Support was provided in part by the IDeA Networks of
Biomedical Research Excellence (INBRE) Program of the National
Center for Research Resources, (P20 RR-16460) to JHH and GPM, the
Summer Undergraduate Research Fellowship sponsored by the
Biochemistry and Molecular Biology Department at UAMS to JHH
and GPM, a bridging grant from the UAMS to GPM. Support was also
provided for GB, in part, by NIH, grant R21ES019684 and the Arkansas
Tobacco Settlement Proceeds Act of 2000.
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