Communication Differential Mechanisms of Cytochrome P450 Inhibition and Activation by a-Naphthoflavone* (Received for publication, November 21, 1995) Aditya P, Koleyt, Jeroen T.M. Buterss, Richard C, Robinsons, Allen Markowitz’, and Fred K. Friedman! Prom the {Laboratory of Molecular Carcinogenesis, NCI ‘and the SBiomedival Instrumentation and Engincering Program, National Institutes of Heatth, Bethesda, Maryland 20802 ‘The anticareinogenicity of some flavonoids has been attributed to modulation of the cytochrome P450 en- ‘zymes, which metabolize procarcinogens to their aeti- vated forms. However, the mechanism by which fla- vonoids inhibit some 'P450-mediated activities while activating others is a longstanding, intriguing question. ‘We employed flash photolysis to measure carbon mon- oxide binding to P450 as a rapid kinetic technique to probe the interaction of the prototype flavonoid o-naph- thoflavone with human cytochrome P450s 1Al and 3A4, whose benzolaipyrene hydroxylation activities are re- spectively inhibited and stimulated by this compound. ‘This flavonoid inhibited P450 1A1 binding to benzo- lajpyrene via a classical competitive mechanism. In on- trast, enaphthoflavone stimulated P450 9A4 by selec- tively binding and activating an otherwise inactive subpopulation of this P450 and promoting benzo- [alpyrene binding to the latter. These data indicate that flavonoids enhance activity by inereasing the pool of active P450 molecules within this P450 macrosystem. Activators in other biological systems may similarly ex- ert their effect by expanding the population of active receptor molecules. Owing to their wide distribution in fruits, vogotables, and rain products, flavonoids are a regularly consumed eompanent of the human diet (1, 2). Inereased consumption of these phy> tochemieals is associated with decreased risk for colon, rectum, ‘and lung cancers (3, 4). Anticareinogenicity in rodents (5,6) has heen attributed to modulation ofthe eytochrome P450 enzymes that metabolize xenobiotic and endogenous compounds, inelud- ing activation of procarcinogens to their earcinogenie forms (7-9). Numerous studios have shown that individual flavonoids may either activate or inhibit P450-mediated activities depend- ing on the target P450 form (10-12). The mechanism of fla vonoid action is unclear yet of intense interest owing to the putative role of dietary flavonoid consumption in P450-med- ‘ated chemical carcinogenesis and the potential for development. of therapeutic flavonoid modulators of specific P&50s. a-Naph- = The costs of publication of this article were defrayed in part bythe ‘payment of page charges. This article must therefore be hereby marked eetcmon sn acordanen wih 18 USC. Seon 129 sey to "To whom correspondence shouldbe addressed: NIH, Bldg. 3, Ran 36-24, Bethesda, MD 20802, "Tel: 901-496-6965, Fax- 901-196-8419; E-mail: ftredshelic.nh.pos. ‘This paper i aaa online at p/w be stannic “Turd or Revenrn Comer, Xt tom ry pe Se thoflavone (ANF! isa protatype Mavonoid whose inhibition of PAbO-mediated activities was first reported over 25 years ago (18) and that has subsequently been used to examine the mech- anism of flavonoid action (14, 15). Most of these studies have assessed the effect of ANF on P450-mediated hydraxylation of the polvevelic aromatic hydrocarbon benzolaipyrene (BP), an ‘environmental pollutant present in cigarette smoke and pol luted air that is carcinogenic in experimental animals (16). ‘The P45O system metabolizes BP to a variety of products including activated metabolites that covalently bind to cellar ‘macromolecules and initiate carcinogenesis (17, 18). Among the human P450s primarily responsible for metabolizing BP (18) are P450s 344 and 1A. P450 3A4 is a major liver PASO that also metabolizes many important drugs (19), whereas P40 AL is normally undeteetable in human liver but present in jung (20), where itis 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 metabo- lism by both human liver mierosomal PA5Os (26-29) and PA50 AA (14, 25, 29, 30) Classical mechaninms such as competitive inhibition ar noncompetitive modulation of substrate binding have usually been invoked to explain such inhibitory or stim- latory effets (14,15). In order to clarify the mechanism of the opposing effects of ANF on Ps 244 and 1A1, we sought to identify differential tmode af interaction af ANF with these Pa5Os. To address this ‘question we applied a rapid kinetic technique, CO binding to PA50 after laser induced fash photolysis of the P450 heme Fe-CO bond, as a sensitive probe of P46O conformation and dynamics. This unique approach contrasts with classical static ‘measurements, which reflec the average properties af a mac rosytem, and successfully revealed subtle and otherwise unde- tectabie mechanistic details for several hemeprateins (31). ‘Thus, a higher rate of CO binding indicates « wide ligand access channel and/or flexible protein, whereas a lower rate suggests a restricted access channel andlor rigid protein con- formation. This kinetic approach was previously used to define 'P450-substrate interactions using both liver microsomes (32, £83) and individual P450s (34, 95 MATERIALS AND METHODS Human P&50s 3A4 and TA1—These P4508 were expressed in SES {nsec cells sing recombinant baculoviruses (96,87) and prepared for flash photolysis os described (34, 35 (CO Flash Photaysi Reactions wore carried out using 0.8 neal of PASO 344 (corresponding to2.2 mga cell homogenate protein) o 0.3 fumalial of F480 AL (orrsponding to 1 mga cell homogenate Drain) and 20 yt CO at 23°C. BP or ANP (Sigma) were added ar 120 ma atock solution in dimethy sulfide? to yield a final exneen tration of 20 yar, beens initial experiments showed that this eoacet {ration produced maximal effets on the CO binding kinetics, Where Indicated, ower concentrations of ANF were used. The mixture wes {hen ineubated for 20 min prior o addition of CO. Photodinscaton of the PASO heme Fe-CO complex and monitoring ofO binding kinetin at ‘50 nn were performed a described (32 When both ANF and BP were Pewee carves were stand regards oftheir order of Tata Analrsis~A kinetic diference tethod was applied to kineti- cally distngsa he inl PAS apt fra the al PA8D spies (21 This approach evaluates the difference between the kinetic rales "The abbreviations used are: ANF, e-naphtheflavone: BP, benzo- lalpsrene. 31493150 ‘obtained in the presence and the absence of « PASO effector and thus ‘tlectivly cancels nt the contributions from PASO that donot bind the ‘ffactor. Using ths approach, kinetic parameters for individual effector- specific PASO SAS and TAT species were obtained by least squares fitting of the difference curves to the kinetic dilforenee Equation 1, an aA= Seer Dae where 4 and A, are the absorbance changes obaerved at ine for The rection in the presence andthe abner of efector. nd re the aberbanc changes and Hand are the poco ret order rae ‘stants forthe efter pei PO inthe presen andthe asene of ‘ico, rexpectively. Th, when a ingle PASO spaces perturbed = 2) ths egunon simply ree tas dilerence of to expen terms, oat) 0.020 ois 010 A 0.000 00 02 04 06 08 10 Time (see) 0.006 0.003 6.000 0.003 c 0.000 Difference in 4 absorbance 0003, 0006 00 02 04 06 08 10 ‘Time (see) io. 1. A, llot of BP and ANF on CO binding kinetics of PA50 141 Progress curves inthe absence a) and the presence of BP() and ANE {o) The CO concentration wa 20 yt effector concentration was 20 yt; dS) TAL concentration was 0.3 uml i 0.1 w sodium ‘nller (pH 7.) containing 20% (we) glycerol. The temperature was 25°C. B, difference between traces band in A for BPC, diffrence between trace panda infor ANF. The solid ines ropresent the best fit acurding tothe Kinetic diflerence Bquation 1. Cytochrome P450 Inhibition and Activation by ANF Enzyme Ausay—BP hydroxylation was assayed by measuring Nus- eacent phenolic metabalites (38). The PABO 2A4 and BP concentrations ‘were the same as those used in the flash photolysis experiments, ‘whereas the ANF concentration was 0, 1,5, oF 20 nt The reaction ‘volume was 0.125 mal and included NADPI-eytochrome PASO reductase (9) in fold molar excess over PASD.A Semin anaay was chosen after ‘establishing the linearity of the reaction rate [RESULTS AND DISCUSSION Fig, 1A depicts the time course for CO binding to P50 11 in the absence and the presence of the substrate BP and the inhibitor ANF. These compounds decreased the overall reac- tion rate (as gauged by half-time) in the order ANF (0:20-s), BP (0.15 2), and no addition (0.075 s). However, precise kinetic definition of the effects ofthese agents requires further analy- sss. 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 ofthe individual P45O species, however, ‘ean 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 1B and C) shows the difference curves for the data in Fig. 1A ‘along with the least squares fits to the kinetic difference equa- tion. This procedure yielded ft, and &;, which represent the CO binding rate constants for a effector-specifie PASO 1A1 species in the absence and the presence of the effector, respectively. ‘These results revealed (Table 1) that BP decreased the rate of CO binding to its target PASO LAL species by Sold (from 19.7 to 68 s-*), whereas ANF similarly decelerated CO binding by ‘3.5-fold (from 19.4 to 54 s-*), Because the experiments with BP and ANF yielded similar (p > 0.05) values for ky (19.7 and 19.45) anda, (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 kineties 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) frum those obtained with ANF alone (Table 1. 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 eourse curves for CO binding to P450 3A4 are illus- trated in Fig. 24. 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 1) that BP decreased the rate of CO binding to 4 PA50 SA4 species by 4.5-fold (from 19.1 to 4.2 s-"), whereas ANF decreased the rate ofits target species by 16-fold (from 92.7 02.4 *), Furthermore, the large difference between the ‘tae 1 Kinetic parameters for CO recombination to PASOs 1AL-and 3A inthe presence and the absence of benacalpyrene ‘and a-naphihofavone, obtained. ‘by hint difference analysis Parameters were determined according tothe kinetic difference Equation 1. and Ay are the pseudo first order rate constants, and a, and 0; are the corresponding absorbanevs inthe abwence and the presence a effector, respectively. Means and standard deviations are derived from si least thre experiments, - Pato iter a o & Paso tat Benacal 0127 = 0028 0071 8151 0016 ” oor | coe o ons | 92 0.000 o mm wo BP hydroxylase (pmol min" nmol") ‘Fo. Relationship between BP hydroxylase activity and amount of ANF-bound P450 JA species IL. Kinetic difference ‘Curves were obtained for diferent concentrations of ANF hy subtracting ‘the CO binding eure obtained inthe prasene of ANF alove from that Blaine for BP + ANF toyed diferencia otha Fig 42D, Dilerence analyses were then performed to calculate a, rnflects the amount of ANF-bound species 1. CO binding kinetics and fctivity measurements were performed in the absence (2) and the [presence of 1, § (3), and 20 pat ANF (4), and the eneresponding a, and LP hydroxylase values were pleted. The respective values were O. 0.0041, 0.0087, and 0.0160, and the rospoctive BP hydrorglaso actii- ties were 18, 8.4, 44.5, and 821 pmol min nmol". P480 3A concen tration wae 0. nmol, and BP concentration was 20 jot for all experiments. 0.05) from the ki value (2.4 s~") of ANF-bound species I, confirming that BP targets the latter. ‘These results suguest that ANF stimulates BP metabolism by P50 3A4 (14, 25, 29, 30) by promoting BP binding to P50 AA species II, To examine the relationship between BP bind- ing 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 kinetie difference curves similar to that in Fig. 2D. Ki- netic difference analyses yielded a, values that reflect the amount of ANF-bound species I at each ANF concentration. A plot of 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 activa- tor of BP hydroxylase. For example, the relatively low activity (2.8 pmnol min" nmol) in its absence was markedly enhanced (82.1 pmol min~* nmol~') in the presence of 20 pat ANF. However, 20 wat ANF enhanced BP binding to the total P450 M4 by only 24-old, based on a, values of 0.0068 and 0.0160 for the BP targeted P450 species in the absence and the pres- ‘ence of ANF, respectively (Table I) These results indicate that in the absence of ANF, BP binds P450 BA4 species I, which has little catalytie 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 ANE inhibits P430 1A1 by competitive binding and activates P450 ‘34 by an allosteric enhancement mechanism: PASO species Il, ‘which normally does not bind BP, binds to ANF and undergoes ‘1 confurmational change that promotes BP binding and metab ‘list. 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 toxicilog- ical effects of flavonoids arise from a dual: mode of action, because P450-mediated drug and carcinogen metabolism can be inhibited via classical eompettive inhibition or enhanced by3162 ‘conformational induction of selected PA50 molecules to an ac- tive 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 moleeules. 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 che- rmopreventive agents. 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