(Received for publication, November 21, 1995)
From the The anticarcinogenicity of some flavonoids has
been attributed to modulation of the cytochrome P450 enzymes, which
metabolize procarcinogens to their activated forms. However, the
mechanism by which flavonoids inhibit some P450-mediated activities
while activating others is a longstanding, intriguing question. We
employed flash photolysis to measure carbon monoxide binding to P450 as a rapid kinetic technique to probe the interaction of the prototype flavonoid Owing to their wide distribution in fruits, vegetables, and grain
products, flavonoids are a regularly consumed component of the human
diet (1, 2). Increased consumption of these phytochemicals is
associated with decreased risk for colon, rectum, and lung cancers (3,
4). Anticarcinogenicity in rodents (5, 6) has been attributed to
modulation of the cytochrome P450 enzymes that metabolize xenobiotic
and endogenous compounds, including activation of procarcinogens to
their carcinogenic forms (7-9). Numerous studies have shown that
individual flavonoids may either activate or inhibit P450-mediated
activities depending on the target P450 form (10-12). The mechanism of
flavonoid action is unclear yet of intense interest owing to the
putative role of dietary flavonoid consumption in P450-mediated
chemical carcinogenesis and the potential for development of
therapeutic flavonoid modulators of specific P450s. The P450 system metabolizes BP to a variety of products including
activated metabolites that covalently bind to cellular macromolecules and initiate carcinogenesis (17, 18). Among the human P450s primarily
responsible for metabolizing BP (18) are P450s 3A4 and 1A1. P450 3A4 is
a major liver P450 that also metabolizes many important drugs (19),
whereas P450 1A1 is normally undetectable in human liver but present in
lung (20), where it is induced by cigarette smoking and is associated
with lung cancer (19, 21). ANF inhibits P450 1A1-mediated metabolism of
BP by rat liver microsomal P450s (22, 23) and human P450 1A1 (24, 25),
yet stimulates BP metabolism by both human liver microsomal P450s
(26-29) and P450 3A4 (14, 25, 29, 30). Classical mechanisms such as
competitive inhibition or noncompetitive modulation of substrate
binding have usually been invoked to explain such inhibitory or
stimulatory effects (14, 15).
In order to clarify the mechanism of the opposing effects of ANF on
P450s 3A4 and 1A1, we sought to identify a differential mode of
interaction of ANF with these P450s. To address this question we
applied a rapid kinetic technique, CO binding to P450 after laser
induced flash photolysis of the P450 heme Fe-CO bond, as a sensitive
probe of P450 conformation and dynamics. This unique approach contrasts
with classical static measurements, which reflect the average
properties of a macrosytem, and successfully revealed subtle and
otherwise undetectable mechanistic details for several hemeproteins
(31). Thus, a higher rate of CO binding indicates a wide ligand access
channel and/or a flexible protein, whereas a lower rate suggests a
restricted access channel and/or rigid protein conformation. This
kinetic approach was previously used to define P450-substrate
interactions using both liver microsomes (32, 33) and individual P450s
(34, 35).
These P450s were expressed in SF9
insect cells using recombinant baculoviruses (36, 37) and prepared for
flash photolysis as described (34, 35).
Reactions were carried out using 0.8 nmol/ml of P450 3A4 (corresponding to 2.2 mg/ml cell homogenate
protein) or 0.3 nmol/ml of P450 1A1 (corresponding to 1.8 mg/ml cell
homogenate protein) and 20 µM CO at 23 °C. BP or ANF
(Sigma) were added (from a 20 mM stock
solution in dimethyl sulfoxide) to yield a final concentration of 20 µM, because initial experiments showed that this
concentration produced maximal effects on the CO binding kinetics.
Where indicated, lower concentrations of ANF were used. The mixture was
then incubated for 20 min prior to addition of CO. Photodissociation of
the P450 heme Fe-CO complex and monitoring of CO binding kinetics at
450 nm were performed as described (32). When both ANF and BP were present, identical curves were obtained regardless of their order of
addition.
A kinetic difference method was applied to
kinetically distinguish the individual P450 species from the total P450
species (32). This approach evaluates the difference between the
kinetic profiles obtained in the presence and the absence of a P450
effector and thus effectively cancels out the contributions from P450s that do not bind the effector. Using this approach, kinetic parameters for individual effector-specific P450 3A4 and 1A1 species were obtained
by least squares fitting of the difference curves to the kinetic
difference Equation 1,
Laboratory of Molecular Carcinogenesis,
-naphthoflavone with human cytochrome P450s 1A1 and 3A4,
whose benzo[a]pyrene hydroxylation activities are
respectively inhibited and stimulated by this compound. This flavonoid
inhibited P450 1A1 binding to benzo[a]pyrene via a
classical competitive mechanism. In contrast,
-naphthoflavone
stimulated P450 3A4 by selectively binding and activating an otherwise
inactive subpopulation of this P450 and promoting
benzo[a]pyrene binding to the latter. These data indicate
that flavonoids enhance activity by increasing the pool of active P450
molecules within this P450 macrosystem. Activators in other biological
systems may similarly exert their effect by expanding the population of
active receptor molecules.
-Naphthoflavone
(ANF)1 is a prototype flavonoid whose
inhibition of P450-mediated activities was first reported over 25 years
ago (13) and that has subsequently been used to examine the mechanism
of flavonoid action (14, 15). Most of these studies have assessed the
effect of ANF on P450-mediated hydroxylation of the polycyclic aromatic
hydrocarbon benzo[a]pyrene (BP), an environmental
pollutant present in cigarette smoke and polluted air that is
carcinogenic in experimental animals (16).
Human P450s 3A4 and 1A1
where
(Eq. 1)
At
and
At are the absorbance changes observed at time
t for the reactions in the presence and the absence of
effector. ai
and ai are the absorbance changes, and
ki
and
ki are the pseudo-first order rate constants for the
effector-specific P450 in the presence and the absence of effector,
respectively. Thus, when a single P450 species is perturbed (n = 1), this equation simply reduces to a difference
of two exponential terms.
BP hydroxylation was assayed by measuring fluorescent phenolic metabolites (38). The P450 3A4 and BP concentrations were the same as those used in the flash photolysis experiments, whereas the ANF concentration was 0, 1, 5, or 20 µM. The reaction volume was 0.125 ml and included NADPH-cytochrome P450 reductase (39) in 2-fold molar excess over P450. A 3-min assay was chosen after establishing the linearity of the reaction rate.
Fig. 1A depicts the time course for CO
binding to P450 1A1 in the absence and the presence of the substrate BP
and the inhibitor ANF. These compounds decreased the overall reaction
rate (as gauged by half-time) in the order ANF (0.20 s), BP
(0.15 s), and no addition (0.075 s). However,
precise kinetic definition of the effects of these agents requires
further analysis. We have previously shown that P450s 3A4 (34) and 1A1
(35) are each comprised of multiple, kinetically distinguishable
species that are unresolvable by standard multiexponential analysis.
The kinetics of the individual P450 species, however, can be resolved
using a kinetic difference method (32, 34, 35), which entails analysis
of a curve generated by subtracting the kinetic data in the absence of
effector from that in the presence of effector. The resulting kinetic
difference profile thus reflects the kinetic properties of the
effector-specific P450 species. Fig. 1 (B and C)
shows the difference curves for the data in Fig. 1A along
with the least squares fits to the kinetic difference equation. This
procedure yielded k1 and
k1, which represent the CO
binding rate constants for a effector-specific P450 1A1 species in the
absence and the presence of the effector, respectively. These results
revealed (Table I) that BP decreased the rate of CO
binding to its target P450 1A1 species by 3-fold (from 19.7 to 6.8 s
1), whereas ANF similarly decelerated CO binding by
3.5-fold (from 19.4 to 5.4 s
1). Because the experiments
with BP and ANF yielded similar (p > 0.05) values for
k1 (19.7 and 19.4 s
1) and
a1 (0.0071 and 0.0084), these results indicate
that both compounds interact with same P450 1A1 species.
|
We next assessed the effect of ANF on BP binding to its target P450 1A1 species by measuring the kinetics of P450 1A1 pre-equilibrated with both BP and ANF. The progress curve was identical to (data not shown) and the kinetic parameters were indistinguishable (p > 0.05) from those obtained with ANF alone (Table I). The observation that BP had no effect on the CO binding kinetics when ANF was present, in conjunction with the known metabolism of ANF by this P450 (25), indicates that ANF competitively inhibits BP binding.
Time course curves for CO binding to P450 3A4 are illustrated in Fig.
2A. The addition of either BP or ANF
decelerated the overall rate, although ANF was clearly more potent.
Data analyses by the kinetic difference method (Fig. 2, B
and C) revealed (Table I) that BP decreased the rate of CO
binding to a P450 3A4 species by 4.5-fold (from 19.1 to 4.2 s1), whereas ANF decreased the rate of its target species
by 16-fold (from 37.7 to 2.4 s
1). Furthermore, the large
difference between the corresponding k1 values
(19.1 and 37.7 s
1, p < 0.05) clearly
shows that BP and ANF interact with different P450 3A4 species, termed
species I and II, respectively. In addition, the
a1 values reflect the amounts of these two
species and show that the ANF-reactive species is more predominant than
the BP-reactive species (a1 = 0.0186 and 0.0068, respectively).
The next question is: because BP and ANF interact with different P450
3A4 species, how does ANF activate BP metabolism? To address this
question we measured CO binding in the presence of both BP and ANF
(Fig. 2A, d). The resulting CO binding rate was less than that in the presence of BP alone but greater than that observed for ANF alone. Thus BP, in contrast to its decelerating effect
on species I, increased the rate of the ANF-bound species II.
Application of the kinetic difference method by subtraction of the
curve in the presence of ANF alone (Fig. 2A, c)
from that in the presence of both BP and ANF (Fig. 2A,
d) yielded a difference curve (Fig. 2D)
representing BP binding to a single ANF-bound P450 species (Table I).
The results firstly show that BP increased the CO binding rate of its
target ANF-bound P450 3A4 species II by 4-fold (from 2.2 to 9.4 s1), in contrast to the BP-induced rate deceleration for
species I. The k1 value (2.2 s
1)
is furthermore indistinguishable (p > 0.05) from the
k1
value (2.4 s
1) of ANF-bound species II, confirming that BP targets
the latter.
These results suggest that ANF stimulates BP metabolism by P450 3A4
(14, 25, 29, 30) by promoting BP binding to P450 3A4 species II. To
examine the relationship between BP binding and metabolism, we measured
both CO binding kinetics and BP hydroxylation at different levels of
ANF-bound species II by varying the ANF concentration. Kinetics
experiments were performed in the presence of BP + ANF and ANF alone to
yield kinetic difference curves similar to that in Fig. 2D.
Kinetic difference analyses yielded a1 values that reflect the amount of ANF-bound species II at each ANF
concentration. A plot of a1 versus BP
hydroxylase activity (Fig. 3) revealed that these are
well correlated (r = 0.98) and establishes a functional link between BP binding to species II and BP metabolism. The data further show that ANF is an exceptionally potent activator of BP
hydroxylase. For example, the relatively low activity (1.8 pmol
min1 nmol
1) in its absence was markedly
enhanced (82.1 pmol min
1 nmol
1) in the
presence of 20 µM ANF. However, 20 µM ANF
enhanced BP binding to the total P450 3A4 by only 2.4-fold, based on
a1 values of 0.0068 and 0.0160 for the BP
targeted P450 species in the absence and the presence of ANF,
respectively (Table I). These results indicate that in the absence of
ANF, BP binds P450 3A4 species I, which has little catalytic activity.
In contrast, in the presence of ANF, BP binds the highly active
ANF-bound species II.
The results are summarized in Fig. 4, which shows that
ANF inhibits P450 1A1 by competitive binding and activates P450 3A4 by
an allosteric enhancement mechanism: P450 species II, which normally
does not bind BP, binds to ANF and undergoes a conformational change
that promotes BP binding and metabolism. This model thus differs from
the classical allosteric mechanism, which does not account for the
conformational heterogeneity of proteins.
These data indicate that the pharmacological and toxicological effects of flavonoids arise from a dual mode of action, because P450-mediated drug and carcinogen metabolism can be inhibited via classical competitive inhibition or enhanced by conformational induction of selected P450 molecules to an active form. The latter mechanism is similar to the recently described activation of receptors by agonists (40, 41) and may prove to be generally applicable in protein interactions with small molecules. Such elucidation of the diverse mechanisms of flavonoid action enhances our understanding of the role of dietary flavonoids in modulating P450-mediated reactions and may aid in the identification and design of flavonoids as chemopreventive agents.