Chem. Res. Toxicol. 1993,6, 872-879
872
Inhibition and Inactivation of Murine Hepatic Ethoxy- and Pentoxyresorufin O-Dealkylase by Naturally Occurring Coumarins Yingna Cai,? Daniel Bennett,? Raghunathan V. Nair,t Oluna Ceska,t Mike. J. Ashwood-Smith,t and John DiGiovanni*pt University of Texas, M . D. Anderson Cancer Center, Science Park-Research Division, Department of Carcinogenesis, P.O.Box 389, Smithville, Texas 78957, and Department of Biology, University of Victoria, Victoria, British Columbia V8W 2Y2, Canada Received July 7, 199P
The present study was designed to evaluate the effects of a series of natural coumarins on ethoxyresorufin O-dealkylase (EROD) and pentoxyresorufin O-dealkylase (PROD) activities in vitro using hepatic tissues from SENCAR mice. Fifteen different coumarins were examined for potential modulating activities. Several naturally occurring coumarins, found in the human diet, were effective inhibitors of hepatic EROD activity in vitro, including coriandrin, bergamottin, isoimperatorin, and ostruthin. Notably, coriandrin and bergamottin were approximately as potent as 7,8-benzoflavone, a relatively selective inhibitor of cytochrome P450 1Al. Several naturally occurring coumarins were also potent inhibitors of hepatic PROD activity, including imperatorin, bergamottin, isopimpinellin, and angelicin. Kinetic studies of the type of inhibition revealed that these compounds inhibited hepatic EROD and PROD activity by a variety of modes rather than by a uniform one. Furthermore, experiments using a two-stage incubation assay revealed that coriandrin, imperatorin, ostruthin, and several other natural coumarins inactivated hepatic EROD activity (i.e., predominantly cytochrome P450 1Al-mediated) and that isopimpinellin inactivated hepatic PROD activity (Le., predominantly cytochrome P450 2B1-mediated). Finally, the results indicate that some coumarins had selective inhibitory effects for EROD vs PROD and preliminary analyses suggested a possible structural basis for the observed differences. T h e current data suggest that certain naturally occurring coumarins, to which humans are exposed in the diet, are potent modulators of cytochrome P450. Furthermore, these compounds may be capable of influencing the metabolic activation of other xenobiotics, including chemical carcinogens. Chart I
Introduction Coumarins are widely distributed in nature and are found in all parts of plants (I). These compounds are especially common in grasses, orchids, citrus fruits, and legumes (I, 2). Being so abundant in nature, coumarins make up an important part of the human diet. Based on chemical structure (see Chart I), they can be broadly classified as (a) simple coumarins (e.g., coumarin, 11, (b) furanocoumarins of the linear (e.g., imperatorin, 9) or angular (e.g., angelicin, 15) type, and (c) pyranocoumarins of the linear (e.g., xanthyletin, 14) or angular (e.g., seselin, 16) type (I). Simple coumarins are very widely distributed in the plant kingdom ( I ) . Interestingly, citrus oils, in particular, contain abundant amounts of both simple as well as furanocoumarins (3). Humans are also exposed to furanocoumarins (e.g., bergapten, 6 and xanthotoxin, 7) in umbelliferous vegetables such as parsnips, celery, and parsley in substantial amounts (ref 4 and references therein). For example, parsnip root reportedly contains as much as 40 mg/kg of certain linear furanocoumarins such as psoralen, xanthotoxin (7), and bergapten (6) which are not destroyed by normal cooking procedures (boiling or microwave) (4). In addition, in certain countries (e.g., China, India, and Mexico) and in certain geographical areas
4. 5-Geranoxy-7-m&oxyoumnrin
Rz= O V \ y
5. Ostruthin RI, R,=H R,=OH R?=
11. Bergamonin R,=H;
'"'(
12. coriandrin
15. Angelicin
* Author to whom correspondence and reprint requests should be addressed. t University of Texas. t University of Victoria. Abstract published in Advance ACS Abstracts, October 15, 1993.
10. himperatorin RI=H;
13. Dihydrocoriandrin
16. Seselin
14. Xanthyletin
17.7.8-Benzoflavone
within the United States (e.g., the Southwest) fresh coriander leaves (alsoknown as cilantro or Chinese parsley) are used extensively. The cilantro leaves are used in soups,
Q893-228~/93/21Q6-Q872$04.00/0 0 1993 American Chemical Society
Inhibition of Monooxygenase by Natural Coumarins chutneys, and sauces and for flavoring curries and even wine. Recently, several furoisocoumarins (e.g., coriandrin, 12,and dihydrocoriandrin, 13)have been identified in fresh coriander ( 5 , 6 )and thus humans can be exposed to these compounds regularly in the diet. A limited number of studies (7-9) have examined the potential inhibitory effects of coumarins against chemically-induced cancer in rodents. Coumarin (1)and 4methylcoumarin (not shown) when administered orally were moderately effective inhibitors of 7,12-dimethylbenz[alanthracene (DMBAY induced mammary tumors in the rat and showed inhibitory activity for benzo[alpyrene (B[a] P) induced neoplasia of the mouse forestomach, while limettin (2)was a less effective inhibitor of DMBA-induced mammary tumors and was inactive for B[alP-induced neoplasia of the mouse forestomach. In general, simple coumarins having polar substitutents were found to be relatively poor inhibitors of tumorigenesis in these model systems. Sparnins and Wattenberg (10) demonstrated that coumarin (1) enhanced forestomach glutathione S-transferase (GST) activity and sulfhydryl levels following 2 weeks of dietary administration to mice. A good correlation between ability to elevate GST and inhibit B[a]P-induced forestomach tumors was noted for a series of compounds (10). However, it should be stressed that little else is known about this widely distributed class of naturally occurring chemicals, especiallystructure-activity relationships and potential mechanisms by which coumarins may inhibit the carcinogenic process. The inhibitory effects of several 8-acyl-7-hydroxycoumarine on 3-methylcholanthrene (MC) induced rat hepatic aryl hydrocarbon hydroxylase (AHH) have been reported (11). Imperatorin (9) and several derivatives have been reported to both induce and inhibit drug-metabolizing enzymes (12). Notably, imperatorin (9) was an effective antimutagen for 2-aminoanthracene and B[a]P in Salmonella typhimurium in the presence of a hepatic 9OOOg supernatant (SS)activating system (13). Several other naturally occurring coumarins also displayed antimutagenic activity to varying degrees in this system, including coumarin (l), umbelliferone (not shown), psoralen (not shown), and osthol (not shown). Bergapten (6) and xanthotoxin (71, when added in vitro, inhibited hepatic microsomal 7-ethoxycoumarin 0-deethylase (ECD), B[a]P hydroxylase, aminopyrene N-demethylase (AP-N-demethylase), and hexobarbital hydroxylase (HB-hydroxylase) (14,15). Xanthotoxin (7)was reportedly a potent inhibitor of the in vivo phase I metabolism of phenytoin, hexobarbital, caffeine, and theophylline (16, 17). This furanocoumarin (7) was also found to be an inducer of hepatic ECD, AHH, and ethylmorphine deethylase, when administered orally to rats a t relatively low doses over several days (18,19). Recently, it has been demonstrated that xanthotoxin (7) can be metabolically activated to intermediates that bind covalently to liver microsomal protein and that the bioactivation of xanthotoxin (7) led to the inactivation of cytochrome P450 through covalent modification of the apoprotein (14, 20, 21). Finally, 1 Abbreviations: AHH, aryl hydrocarbon hydroxylase; AP,aminopyrene; B[alP, benzo[alpyrene; 7,8-BF, 7,8-benzoflavone; DMBA, 7,12dimethylbenz[a]anthracene; ECD, %ethoxycoumarin0-deallrylase;EROD, ethoxyresorufii 0-dealkylase; G-6-P, glucose 6-phosphate; G-6-PD, glucose-&phosphatedehydrogenase;GST, glutathione S-transferase; HB, hexobarbital; MC, 3-methylcholanthrene; PAH, polycyclic aromatic hydrocarbon; PROD, pentoxyresorufiin0-dealkylase; Pb, phenobarbital; Se, 9000gsupernatant, TBPOBOP, 1,4-bis[[2-(3,5-dibromopyridyl)loxylbenzene.
Chem. Res. Toxicol., Vol. 6, No. 6, 1993 873 coriandrin (12) has been shown to be metabolized very rapidly by rat liver microsomal preparations (6). All of these data demonstrate the likelihood that naturally occurring coumarins ingested by man may have marked effects on the enzyme systems involved in metabolizing xenobiotics, including polycyclic aromatic hydrocarbons (PAHs) and other carcinogens. In the present study, a series of naturally occurring coumarins including simple coumarins, linear and angular furanocoumarins, furoisocoumarins,and linear and angular pyranocoumarins were investigated for their effects on murine hepatic ethoxyresorufin 0-dealkylase (EROD)and pentoxyresorufin 0-dealkylase (PROD) activities. These two compounds were used because they represent relatively selective substrates for cytochrome P450 1 A l and P450 2B1 activities, respectively (22-25). The present results show that several natural coumarins had potent inhibitory effects on either EROD or PROD activities or both. Further examination of selected coumarins revealed their ability to inactivate EROD or PROD activity depending on the compound. The results demonstrate that naturally occurring coumarins, especially the linear furanocoumarin or furanoisocoumarin type, are potent inhibitors of in vitro microsomal monoxygenases and that they may represent a general class of mechanism-based inactivators of cytochrome@)P450 to which humans are exposed in the diet.
Experimental Procedures Materials. Imperatorin (9) was purchased from Indofine Chemical Co. (BelleMead, NJ). Xanthotoxin (7),coumarin (l), limettin (2), and 7,8-benzoflavone (7,8-BF)(17)were purchased from Aldrich ChemicalCo. (Milwaukee,WI). Isopimpinellin (8), isoimperatorin (lo),bergapten (6),&propylene, 6-methoxycoumarin (3),and angelicin (15)were supplied by Dr. Wayne Ivie, Agricultural Research Service USDA, College Station, TX. Coriandrin (12) and dihydrocoriandrin (13) were obtained as previously described (5,6). 5-Geranoxy-7-methoxycoumarin (4), ostruthin (5),bergamottin (111,xanthyletin (14),andseselin (16) were obtained from Dr. Warren Steck,National Research Council of Canada, Prairie Regional Laboratory, Saaskatoon, Saskatchewan. Ethoxyresorufii,7-pentoxyresorufii,glucose 6-phosphate (G-6-P), glucose-6-phosphate dehydrogenase (Gg-PD), and NADP were obtained from SigmaChemical Co. (St. Louis, MO). MC was purchased from Eastman Kodak Co. (Rochester, NY) 1,4-Bis[[2-(3,5-dibromopyridyl)]oxylbenzene(TBPOBOP) (26) was a generousgift from Dr. Stephen Safe,TexasA&MUniversity, and was used as a phenobarbital (Pb)-like inducer. The purity of all the coumarins was checked by melting point and HPLC analysis. All chemicals used in the current study were more than 96% pure as judged by HPLC. Other reagents were obtained commercially and were of the highest purity demand necessary. The coumarins, especially the furano- and pyranocoumarins as well as MC used in the present study should be considered carcinogens and handled with extreme care. Microsomal Preparation. Female SENCAR mice (purchased from NCI, Frederick, MD)at 7-9 weeks of age were treated with MC dissolved in olive oil (80mg/kg of body weight, single dose daily, ip) for two consecutive days or with TBPOBOP dissolvedin olive oil (5.8 mg/kg of body weight, single dose daily, ip) for three consecutive days. The mice were sacrificed 24 h after the last treatment and their livers were removed and homogenized in 0.05M Tris4.25 M sucrose buffer (pH 7.5) using a Polytron PT 10 homogenizer (setting 6, 45 s). Microsomal fractions were obtained by centrifuging the whole homogenate at 9000g for 30 min and then centrifuging the Se at 105000g for 60 min. Microsomal pellets were resuspended in 0.05 M Tris0.25 M sucrose buffer (pH 7.5) (-2 of mg protein/mL). Protein concentrations were determined by the method of Lowry (27).
Cui et al.
874 Chem. Res. Toxicol., Vol. 6, No. 6, 1993
EROD and PROD Assays. The respectiveenzyme activities were determined in reaction mixtures (1 mL total volume) containing MC-induced microsomes (0.03 mg of protein) or TBPOBOP-induced microsomes (0.05 mg of protein), ethoxyresorufh or 7-pentoxyresorufin(5pM), an NADPH-generating system (0.336 mM NADP+,0.5 mM G-6-P,0.15 mM MgC12, and 1 unit of G-&PD), 0.05 M Tris buffer (pH 7.5), and various concentrationsof coumarins. After incubation for 10 min at 37 OC, the reaction was terminated by the addition of 2.5 mL of methanol. The formationof product (resorufin)was determined in a Hitachi Model F-2000 fluorescence spectrophotometer (hx = 550 nm, X,. = 585 nm). Control incubations did not contain the coumarins. Percent inhibition of the enzyme activity at various concentrations of coumarins was calculated, and IC% values were obtained from 50% inhibition of enzyme activity by interpolation. IC%values represent an average of two separate experiments. Kinetics of Inhibition of EROD and PROD by Selected Coumarins. Enzyme assays were as described in the preceding
section. ApparentKivalues were calculated by using the following equations (28): (i) for competitive inhibition, Ki = Km[I]/(Km’ -K& (ii)for noncompetitive inhibition,Ki = v-’[Il/u--v-’; and (iii) for mixed inhibition, Ki = v-’Km[II/Km’v- v-’Km and K( = v,’[I]/v, ,‘,,Y where K,, v- and K,’, ‘-v were obtained in the absence and in the presence of inhibitor, respectively, and [I] is the concentration of the inhibitor. Inactivation of EROD and PROD by SelectedCoumarins. A two-stageincubation procedure was used to examine the time-
dependentinactivation of EROD and PROD (29). The first stage contained 0.05 M Tris, (pH 7.5), MC-inducedmicrosomes (3 mg of protein/mL), or TBPOBOP-induced microsomes (4 mg of protein/mL), various concentrations of selected coumarins, and an NADPH-generating system as described above. At various times during the incubation (37 OC), an aliquot of the first-stage incubation mixture was diluted 100-fold into a second-stage incubation (37 “C) which contained 0.05 M Tris buffer, (pH 7.51, ethoxyresorufin or 7-pentoxyresorufi (10pM),and the NADPHgenerating system. The second-stageincubation was carried out exactlyas described in the preceding section. K* was calculated from the following equation, t 1 / 2 = 0.693/&,&, where t l l z is the half-life for enzyme inactivation (30).
Results Survey of Selected Natural Coumarins for Inhibition of EROD and PROD Activity. A series of naturally occurring coumarins were investigated for their ability to inhibit murine hepatic EROD and PROD activity in vitro (Table I). As shown in Table I, a number of different classes of coumarins were represented in this study, including simple coumarins (2-5), linear furanocoumarins (6-111,linear furoisocoumarins (12 and 13), linear pyranocoumarin (141,and angular furano- (15)and pyranocoumarins (16). Shown in Table I are the ICm’s determined over a broad concentration range for each compound (5 X lo4 to 5 X 10-10M). In general, the linear furanocoumarins were the most effective inhibitors of EROD activity of which bergamottin (1 1) and coriandrin (12) were the most potent. These two compounds, especially the former, possessed 1C50’s on the order of that obtained with 7,8-BF in our in vitro assay. 7,8-BF is a potent, well-known inhibitor of cytochrome P450lAl(31, 32). One exception to the generality that linear furanocoumarins were the most effective inhibitors of EROD activity was ostruthin (5),a simple coumarin with a geranyl side chain at Ce, which was the fifth most effective inhibitor (based on ICm values) of EROD activity analyzed in the current study. Analysis of the same set of compounds for their ability to inhibit murine hepatic PROD activity induced by
Table I. ICWValues for Selected Naturally Occurring Coumarins and 7,8-BF on Mouse Hepatic EROD and PROD Enzyme Activity.
monooxygenase activity EROD PROD
coumarins 11, bergamottin 17,7,8-BFc 12, coriandrin 10, isoimperatorin 5, ostruthin 8, isopimpinellin 9, imperatorin 6, bergapten 15, angelicin 13, dihydrocoriandrin 7, xanthotoxin 16, seselin
1.24 x i t 7 2 x i t 7 2.45 X le7 1.82 X 106 8.10 X le7 1.87 X 10-6 1.40 X 10-8 1.3 X 10-8
1.50 X 10-8 1.9x10-8
2.32 X 10-8 2.32 X 10-8 5X10-8 5.64 X 10-8 9.62 X 10-8 6.73 X 106 3,5-propylene-6-methoxycoumarin~ 7.86 X 106 2, limettin 210-46 14, xanthyletin L 10-4 4,5-geranoxy-7-methoxycoumarin L1o-L
1.3 X 10-8 4x10-7
5.44 X 10-8 1.5 X 10-8 8X1t7 1.24 X 106 1.3 X 10-8 7.7 X 10-8 1.35X 1od 8.58 X 106 2.08 X 106 2.4 X 10-8
a EROD and PROD activities were measured in hepatic microsomea from MC- and TBPOBOP-pretreated SENCAR mice, respectively. MC pretreatment led to an -23-fold induction of EROD activity while TBPOBOP led to an 10-fold induction of PROD activity in female SENCAR mice. Values in the table represent an average of two separate experiments. Similar results were obtained in both experiments. Concentrations giving 50% inhibition of enzyme activitywere greater than or equal to 1X 1o-LM. c Compoundswhich are not naturally occurring.
-
TBPOBOP revealed some interesting results (again see Table I). Imperatorin (9) was the most potent inhibitor (IC50 = 5.4 X lo8M) of PROD activity in vitro, although several other coumarins were also effective inhibitors. Bergamottin (11) also was an effective inhibitor of mouse hepatic PROD activity as well as being one of the most potent inhibitors of EROD activity within the series of compounds tested. Several other compounds had notable inhibitory activity toward PROD including isopimpinellin (8)(IC%, 4 X M), angelicin (15)(IC60 = 8 X le7 M), isoimperatorin (lo), ostruthin (51, bergapten (6), and xanthotoxin (7),all of which had ICm’s of -1 X 10-8 M. Kinetics of Inhibition of EROD and PROD Activities by Selected Coumarins. Seven of the coumarins that inhibited EROD activity were further examined for their mode or type of inhibition. 7,8-BF was again included as a known control compound in these studies. 7,8-BF was reported previously to be a noncompetitive inhibitor of AHH activity in hepatic microsomes from MC-induced rats (32). For the current studies, various concentration of substrate were examined at a single inhibitor concentration. Lineweaver-Burk plots were then generated from the resulting data sets. The results of these analyses are summarized in Figure 1and Table 11. Figure 1shows the results with imperatorin (9) (panel A), bergamottin (11) (panel B), and coriandrin (12) (panel C). These three compounds represented examples of competitive, noncompetitive, and mixed inhibitors. The results with all compounds are summarized in Table 11. In this regard, dihydrocoriandrin (13),xanthotoxin (7),and imperatorin (9)were found to be competitive inhibitors; 7,8-BF (17) and bergamottin (1 1) were found to be noncompetitive inhibitors; and coriandrin (121,isoimperatorin (lo), and ostruthin (5)were found to be mixed inhibitors. Note that the Ki values determined from this analysis were, in some cases, significantly lower than the ICm’s determined in Table I. A similar analysis of mode of inhibition with a limited number of compounds was also performed for PROD.
Chem. Res. Toxicol., Vol. 6, No. 6, 1993 875
Inhibition of Monooxygenase by Natural Coumarins
Table 111. Ki Values for Selected Naturally Occurring Coumarins on Mouse Hepatic PROD Activity in Vitro. ICEOW] Ki[Ml kinetic type compd 9,imperatorin 5.44 X 10-8 4.07 X 10-8 noncompetitive 4.32 X 10-8 noncompetitive 11, bergamottin 2 X 10-7 1.92 X lo-' competitive 8, isopimpinellin 4 X lo-' 5, ostruthin 1.3 X 10-6 3.44 X lo-' noncompetitive "Values in the table represent an average of two separate experiments. Similar results were obtained in both experiments. 1101
I
100
90 80 70 60 50
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-3
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0
6
9
12
7
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00
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1
20
-
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.
30
1101
1
70
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1
60 50
,
,
,
,
,
10
0 0.0
1 .o
2.0
3.0
2.0
3.0
. ..
-6
-3
0
3 6 1 W I (PM)
9
12
90
Figure 1. Lineweaver-Burk plots showing the inhibition of EROD activity by imperatorin (A), bergamottin (B), and coriandrin (C). The incubation mixtures contained MC-induced microsomes (0.03mg of protein), an NADPH-generating system, and EROD (0.2-2 pM) in the absence (B) or the presence (e) of 0.5 pM imperatorin (A), 1 pM bergamottin (B),or 0.5 pM coriandrin (C), respectively, in a final volume of 1 mL. The reaction mixtures were incubated at 37 "C for 10 min. Each data point represents an average of two separate experiments. The units of reaction velocity (V)are pmol/min per mg of protein. Table 11. Ki Values for Selected Naturally Occurring Coumarins and 7,8-BF on Mouse Hepatic EROD Activity in Vitro.
compds
ICs0 (M)
11,bergamottin 17, 7,8-BFb 12,coriandrin
1.24 X lo-' 2.45 X 10-7 8.1 x 10-7
10,isoimperatorin
1.4 X 10-8
6, ostruthin
1.50 X 10-8
9,imperatorin 2.32 x 10-8 18,dihydrocoriandrin 5.64 X 10-8 7,xanthotoxin 9.63 X 10-8
Ki (M) 2.83 X lo-' 5.52 X l@ 1.05 X 10-7(Ki) 2.57 X le' (Ki') 1.36 X 10-7 (Ki) 6.90 X lo-' (Ki') 1.21 X 10-1 (KJ 1.86 X 10-8 (Ki') 1.05 X lo-' 1.58 X 10-8 7.58 X lo-'
100
kinetic type noncompetitive noncompetitive mixed mixed
mixed competitive competitive competitive
OValue in the table represent an average of two separate experiments. Similar results were obtained in both experiments. * Not naturally occurring. Isopimpinellin (8) was found to be a competitive inhibitor whereas, imperatorin (9) and bergamottin (11) were found to be noncompetitive inhibitors. The results of these experiments are summarized in Table 111. Note again that the Ki value for bergamottin (11) determined from this analysis was significantly lower than the ICs0 determined in Table I.
80 70 Bo
50
40 30
"
I
I
0.0
1 .o
~.
Time [min]
Figure 2. Inactivation of EROD activity by coriandrin (A), xanthotoxin (B), and imperatorin (C). First-stage incubations were performed as described under Experimental Procedures for the time indicated. Control incubations containing the highest concentration of each coumarin tested but lacking an NAPPHgenerating system ( 0 )showed no inactivation of EROD activity similar to those incubations containing an NADPH-generating system but lacking coumarin (A).Panel A, coriandrin at 2 pM (+), 5 pM (B), 25 pM (XI, and 50 pM (e);panel B, xanthotoxin at 50 pM (+), 100 pM (U), and 200 pM (X); panel C, imperatorin at 5 pM (+), 50 rM (B), and 100 pM ( X ) . Each data point represents an average of two separate experiments.
Inactivation of Microsomal EROD and PROD by SelectedCoumarins. A two-stage incubation procedure (29)was utilized to examine the ability of selected natural coumarins to inactivate the enzymatic activities associated with murine hepatic microsomalEROD andPROD. These results are summarized in Figures 2 and 3 and Table IV. Six compounds were examined for their abilityto inactivate EROD activity. Figure 2 shows that imperatorin (9), coriandrin (12), and xanthotoxin (7) all possessed the ability to inactivate microsomal EROD activity. Furthermore, ostruthin (5) effectively inactivated EROD activity over a concentration range of 10-100 bM. The ability of these four natural coumarins to inactivate
876 Chem. Res. Toxicol., Vol. 6, No. 6, 1993
Cai et al.
m
..-Cm
aE
ap 0.0
1 .o
2.0
Time [min]
Figure 3. Inactivation of PROD activity by isopimpinellin.Firststage incubation was conducted as described under Experimental Procedure for the times indicated. Control incubationcontaining the highest concentration of isopimpinellin but lacking an NADPH-generatingsystem (0) showed no inactivation of PROD activity similar to those incubations containing an NADPHgenerating system but lacking isopimpinellin (A).Isopimpinellin, 10 pM (+), 20 pM (w), 40 rM (X), 80 pM (+). Each data point came from an average of two separate experiments. Table IV. Summary of Results from Two-Stage
Incubations To Examine Inactivation of Hepatic Microsomal EROD or PROD Activity. KhCt -
compd
EROD
5, ostruthin 9, imperatorin 7, xanthotoxin 12, coriandrin 8, isopimpinellin 11, bergamottin 13, dihydrocoriandrin
0.642 0.280 0.279 0.223 0.177 d e
value (min-l) PROD NDb C
ND ND 1.019 C
ND
aValues in the table represent an average of two separate experiments. Similar results were obtained in both experiments. b Not determined. Did not inactivate PROD activityat overlapping concentrations. Did not inactivate EROD or PROD at an initial concentration of 50 or 25pM, respectively. e Did not inactivateEROD activity at concentrations up to 120 pM.
microsomal EROD activity was dependent on the concentration, time, and NADPH level (see again Figure 2, panels A, B, and C). No inactivation of EROD activity was detectable in the absence of either the coumarins or NADPH [the latter being determined a t the highest concentration of each coumarin tested (see Figure 2)l. The kinetics of inactivation of microsomal EROD activity by imperatorin (9), coriandrin (12), ostruthin (5), and xanthotoxin (7) were pseudo-first-order since inactivation rates were linear for only -2 min. The inactivation of EROD activity by these coumarins also obeyed saturation kinetics (data not shown). As shown in Figure 2, coriandrin (12), xanthotoxin (71, and imperatorin (9) led to a 30%-45% loss of EROD activity over the 2-minute period a t the highest concentrations used. Ostruthin (5) led to a somewhat greater inactivation (-55 % loss of EROD activity over the 2-min period) a t the highest concentration used (data not shown). The Kinactvalues in Table IV for EROD were similar for all coumarins examined (0.1770.280 min-l) except ostruthin (5) whose Kinactwas 0.642 min-l. Notably, neither bergamottin (11) nor dihydrocoriandrin (13) had the ability to inactivate microsomal EROD activity a t concentrations up to 50 or 120 pM, respectively. Two-stage incubation experiments were also conducted to examine potential inactivation of PROD activity by three coumarins; imperatorin (9), bergamottin (111, and isopimpinellin (8). The results shown in Figure 3 and Table IV indicated that isopimpinellin (8) inactivated
PROD activity whereas the other compounds did not. The inactivation of microsomal PROD activity by isopimpinellin was again concentration-, time-, and NADPHdependent (see again Figure 3), similar to the results for the coumarins that inactivated microsomal EROD. The inactivation of PROD by isopimpinellin (8) also appeared to be linear for only -2 min and followed pseudo-firstorder kinetics. The Khd for PROD activity with isopimpinellin (8) (1.019 min-l) was significantly higher than that observed with any of the coumarins that inactivated microsomal EROD activity. The inability of imperatorin (9) to inactivate microsomal PROD is interesting and demonstrates the selectivity of this coumarin for inactivation of microsomal EROD activity and presumably cytochrome P450 1Al. Furthermore, the -6-fold higher Kinactfor PROD activity vs EROD activity with isopimpinellin (8) demonstrates some selectivity with this compound for inactivating microsomal PROD activity and presumably cytochrome P450 2B1.
Discussion This study was designed to evaluate selected, naturally occurring coumarins for their ability to modulate cytochrome P450-mediated enzyme activities. Note that for the purposes of the present study we have assumed that the predominant cytochrome P450 species mediating EROD activity is cytochrome P450 1 A l and for PROD activity is cytochrome P450 2B1 (22-25). Furthermore, we have assumed that the coumarins are primarily affecting these predominant P450 species in their ability to inhibit the associated enzymatic activity. However, we cannot rule out the possibility that the inhibitory coumarins may affect other cytochrome P450 species that contribute in a minor way to the two enzymatic activities. Cytochrome(s) P450 are major oxidative enzymes that metabolize xenobiotics, including chemical carcinogens; therefore, modulation of these enzymes can dramatically affect toxicity and carcinogenesis. The major findings of this investigation are as follows: (i) coumarins, especially several linear furanocoumarins, were potent inhibitors of cytochrome(s) P450-mediated enzyme activities; (ii) some coumarins had selective inhibitory effects on cytochrome P450 1Al- vs P-450 2B1-mediated enzyme activities; (iii) several coumarins also had the ability to inactivate specific cytochrome(s) P450-mediated enzyme activities; and (iv) certain structural features appeared to influence both inhibition and inactivation of cytochrome(s) P450 by the coumarins examined (see discussion below). To date a number of compounds have been reported to be selective inhibitors of cytochrome(s1 P450 including both reversible and mechanism-based inactivators (reviewed in refs 33 and 34). Early studies by Diamond and Gelboin (35) and by Wiebel et al. (36-38) demonstrated that 7,8-BF could distinguish between AHH induced by P b and MC by strongly inhibiting AHH induced by MC without affecting that induced by Pb. Several studies have reported that 7,8-BF preferentially and reversibly inhibits cytochrome P450 1 A l (reviewed in refs 33 and 39) although this compound can weakly inhibit or even activate other forms of cytochrome P450 (39). The response to 7,8-BF can also be highly species dependent (40). Weibel et al. (41) also reported some structure-activity relationships among both synthetic and natural flavones for their effects on specific types of monooxygenase activities as follows: (i) 5,6- and 7,8-benzoflavone and their more
Inhibition of Monooxygenase by Natural Coumarins
hydrophobic derivatives inhibited MC-induced AHH and increased or did not affect the activity of the constitutive enzyme; (ii) derivatives such as the 4’-hydroxylated benzoflavones decreased to a similar degree the MCinduced and constitutive AHH activities; and (iii) polyhydroxyflavones inhibited the constitutive enzyme more than the MC-induced enzyme. Ellipticine and 9-hydroxyellipticine are additional examples of reversible inhibitors that show specificity toward the P450 1A-type isozymes (42,43).More recently, m-xylene was reported to selectively inhibit cytochrome P450 2B1 activity in rat pulmonary microsomes, but it did not display this selectivity for P450 2B1 in rat liver microsomes (44).(-)-Maackiain acetate, a derivative of the naturally occurring parent compound, (-)-maackiain, is a potent inhibitor of control and Pb-induced rat hepatic AHH (32).A large number of other inhibitors, primarily of the mechanism-based type, have been shown to possess specificity toward certain P450 species (reviewed in refs 33, 34, and 45). As noted in the introduction, several studies have reported inhibition of cytochrome P450 by various coumarin derivatives, including both simple (11,461and more complex coumarins (12,14-17,20,21). In a study of several types of coumarins, only the furano- and pyranocoumarins possessed significant ability to inhibit HB hydroxylase and AP N-demethylase activities in hepatic tissue preparations from control rats (12). In these studies, the presence of a double bond in the furano or pyran0 ring appeared to be essential for the manifestation of inhibitory activity, since the furano- and pyranocoumarin with a double bond on the heterocyclic rings showed significant activity while the other coumarins such as dihydrofuranoand dihydropyranocoumarins did not. Furthermore, imperatorin (9) and isoimperatorin (lo), which possess an isopentenoxyl side chain, were found to elicit the strongest inhibitory potency on both HB hydroxylation and AP N-demethylation. The inhibitory potency decreased as the polarity of the side-chain increased. Xanthyletin (14), which is devoid of any side chains, showed the least activity (12).However, it should be stressed that detailed studies of specificity toward specific cytochrome P450 enzymes have not been reported for furano- and pyranocoumarins. Examination of the data in Table I of our study indicate that linear furanocoumarins with Cg-alkoxy substituents such as isopimpinellin (8), imperatorin (9), and xanthotoxin (7) preferentially inhibited P450 2B1(&40-fold lower IC50 values for inhibition of P450 2B1 vs P450 l A l ) , while linear furanocoumarins lacking Cg-alkoxy1 substituents but having Cr-alkoxy1 substituents such as bergamottin (1 l), isoimperatorin (lo), and bergapten (6) effectively inhibited both P450 1 A l and P450 2B1 activity. Furthermore, the degree of inhibition of P450 2B1 appeared to be influenced by the length of the C9 alkoxy1side chain. For example, imperatorin (91, which has an isopentenoxyl side chain a t Cg, was a more potent inhibitor of P450 2B1 than xanthotoxin (7) and isopimpinellin (8), both of which have a methoxy substituent. The data also suggest that furoisocoumarins possessing an isolactone ring [e.g., coriandrin (12) and dihydrocoriandrin (13)] had preferential inhibition of P450 1Al rather than P450 2B1 (IC50 values for P450 1Al were 2.2- and 20-fold lower, respectively, than for P450 2B1). Comparison of these two compounds also indicates that the furano ring double bond dramatically increased the potency for inhibiting cytochrome P450 1Al. In fact, coriandrin (12) exhibited a
Chem. Res. Toxicol., Vol. 6, No. 6,1993 877
relatively selective inhibition of P450 1Al similar to 7,8BF. The data from kinetic studies (Figure 1and Tables I1 and 111)of these compounds also suggested some structureactivity relationships for the type or mode of inhibition observed. Imperatorin (9) and xanthotoxin (71, which have Cg-alkoxy substituents, were competitive inhibitors of P450 1A1,while bergamottin (1 1) and isoimperatorin (lo),which do not have Cg-alkoxy substituents but have C4-alkoxy substituents, were noncompetitive and mixed inhibitors, respectively, of P450 1Al. These data suggest that the position of alkoxy substituents in linear furanocoumarins may determine, in part, the type of inhibition of P450 1Al observed. The two furoisocoumarins, coriandrin (12) and dihydrocoriandrin (13), also behaved differently. Coriandrin (12) was a mixed inhibitor of P450 1A1, whereas dihydrocoriandrin (13) was a competitive inhibitor, suggesting that coriandrin (12) may bind not only to the substrate binding site but also to an additional site that causes loss of enzyme activity. Interestingly, imperatorin (9) was a noncompetitive inhibitor of P450 2B1 but a competitive inhibitor of P450 1Al. This result may help explain why imperatorin (9) was a more potent inhibitor of P450 2B1 since noncompetitive inhibitors, in general, have stronger inhibitory effects than competitive inhibitors (47). As part of the present study, we examined several of the most potent inhibitors for mechanism-based inactivation of cytochrome(s) P450. Xanthotoxin (7) and bergapten (6) were previously reported to inactivate cytochrome P450 (15,21). On the basis of the analysis of several closely related psoralens, it was concluded in one of these studies that metabolism in the furano ring of both compounds was critical for inactivation of cytochrome P450 (15). Alternatively, studies using radiolabeled 8-methoxypsoralen [xanthotoxin (9)l suggested that two different metabolic pathways could lead to inactivation of cytochrome P450 a furano ring metabolite (epoxide ?) and a demethylated intermediate, both of which covalently bound to cytochrome P450 apoprotein (21).As shown in Figures 2 and 3 and Table IV, several of the more potent inhibitors examined had the ability to inactivate cytochrome P450 (primarily P450 1Al). In our studies, we found that coriandrin (12) inactivated cytochrome P450 1 A l whereas dihydrocoriandrin (13) did not. These data indicate that the furano ring double bond is essential for inactivation of this enzyme by coriandrin (12). Dihydrocoriandrin (13),with a methoxy substituent a t C4, did not inactivate P450 1Al indicating that a metabolic activation pathway involving a demethylated intermediate is not operational for this compound. This latter observation is perhaps due to steric hinderance for dealkylationas a result of proximity of the carbonyl oxygen or due to other structural differences in the two furoisocoumarins. Interestingly, ostruthin (5) also inactivated P450 1Al. This simple coumarin lacks both a furano ring and an alkoxy substituent,suggesting that its mechanism of inactivation may be different than that of the other compounds. Finally, bergamottin (11) did not inactivate either P450 1Al or P450 2B1. The closely related bergapten (6), as noted above, was previously reported to inactivate cytochrome P450; thus, the C4 geranoxyl group in bergamottin (1 1) may sterically hinder metabolism in the furano ring and prevent the inactivation cytochrome P450 1 A l .
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Current studies are underway to explore, in more detail, the mechanism(s) for inactivation of specific P450's by these naturally occurring coumarins. In conclusion, the results presented here demonstrate that certain linear furano- and furoisocoumarins and a t least one substituted, simple coumarin [ostruthin (5)l are potent modulators of cytochrome(s) P450. Since humans are exposed to many of the compounds currently examined, as well as others in the diet, it is important to understand fully their pharmacologic and toxicologic properties. Of particular interest and importance is how such dietary constituents might interact and modulate carcinogen metabolizing enzymes. Recently, we reported that several novel synthetic coumarins inhibited the metabolic activation of DMBA in mouse epidermis and that one derivative inhibited tumor initiation by this PAH (48). Future experiments will examine how certain naturally occurring coumarins can modulate carcinogenesis in this model system.
Acknowledgment. This research was supported by USPHS Grants ES 06014 (J.D.) and UTMDACC Core Grant CA 16672, American Cancer Society Grant FRA375 (J.D.), and NSERC of Canada (M.J.A.-S.). We wish to thank Yolanda C. Valderrama for her excellent secretarial skills in the preparation of the manuscript. A portion of this work was presented a t the 84th Annual Meeting of the American Association for Cancer Research (Abstract 1004) held May 1993 in Orlando, FL.
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