Chem. Res. Toxicol. 1993, 6, 188-196
188
Pharmacodynamics of Cytochrome P450 2B Induction by Phenobarbital, 5-Et hyl-5-phenylhydantoin, and 5-Ethyl-5-phenyloxazolidinedione in the Male Rat Liver or in Cultured Rat Hepatocytes Raymond W. Nims,*ftPeter R. Sinclair,t Jacqueline F. Sinclair,l Paul E. Thomas,$ Collins R. Jones,il Donna W. Mellini,l Jia-Lin Syi,* and Ronald A. Lubett Chemistry Section, Laboratory of Comparative Carcinogenesis, National Cancer Institute, Frederick Cancer Research and Development Center, Frederick, Maryland 21 702, Veterans Administration Medical Center, White River Junction, Vermont 05009, Department of Chemical Biology, College of Pharmacy, Rutgers University, Piscataway, New Jersey 08855-0789, and Biological Carcinogenesis and Development Program, Chemical Synthesis and Analysis Laboratory, and Advanced Scientific Computing Laboratory, Program Resources, Inc./DynCorp, NCI-Frederick Cancer Research and Development Center, Frederick, Maryland 21 702 Received November 12, 1992
The pharmacodynamics of rat hepatic cytochrome P450 2B (P450 2B) induction by phenobarbital (PB) and two structural congeners, dZ-5-ethyl-5-phenylhydantoin(EPH) and dZ-5-ethyl-5-phenyloxazolidinedione (EPO), were investigated. The in vivo induction of P450 2B was probed in F344/NCr rats by measuring immunoreactive hepatic P450 2B1 protein and by assaying the hepatic 16/3-hydroxylation of testosterone and O-dealkylation of (benzyloxy)and pentoxyresorufin. The induction of (benzy1oxy)resorufin O-dealkylation activity was also measured in adult rat hepatocyte cultures exposed to the three xenobiotics. The concentration of xenobiotic at the putative active site in the in vivo studies was approximated by measuring serum total xenobiotic levels, while in the hepatocyte culture studies, the nominal xenobiotic concentration in the culture medium was used. Concentration-dependent induction of P450 2B activities was observed in the in vivo and hepatocyte culture studies. The in vivo ED50 values for P450 2B induction were 110, 100, and -3000 dietary ppm (14 days administration) for PB, EPH, and EPO, respectively. The in vivo EC50 values for P450 2B induction were -9, -6, and 130 pM (total serum) for PB, EPH, and EPO, respectively. In cultured rat hepatocytes, the ED50 values for induction of (benzy1oxy)resorufin O-dealkylation activity were 14.5, 14.2, and 108 pM for PB, EPH, and EPO, respectively. These data indicate that pharmacodynamic results obtained with cultured hepatocytes represent a good qualitative and quantitative approximation of the in vivo hepatic responses in male rats caused by PB-type inducers. In both systems, E P H and P B were approximately equivalent in potency for P450 2B induction, while EPO was roughly an order of magnitude less potent.
- -
-
Introduction While receptor-mediated mechanisms for the induction of cytochrome P450 1 A l (P450 1Al)' and 1A2 by aromatic hydrocarbons (1,2 ) ,and more recently for the induction of isozymes of the P450 4A subfamily by peroxisome proliferating agents (3, 4 ) , have been characterized, the mediation of a receptor in the induction of isozymes of the P450 2B subfamily by phenobarbital (PB)-type inducers has yet to be demonstrated (2,5,6).The existence of such receptor mediation would be supported by the demonstration of a clear set of structural requirements for P450 2B-type inducing ability, as well as by the demonstration
* To whom correspondence should be addressed at Building 538,Room 205E, Frederick Cancer Research and Development Center, Frederick, MD 21702. National Cancer Institute. * VA Medical Center. Rutgers University. 1 PRIiDynCorp, Biological Carcinogenesisand Development Program. 1 PRI/DynCorp, Chemical Synthesis and Analysis Laboratory. PRIiDynCorp, Advanced Scientific Computing Laboratory. Abbreviations: P450 1A1, cytochrome P450 isozyme 1Al; P450 2B, cytochromeP450subfamily2B;EPH,dl-5-ethyl-5-phenylhydantoin; EPO, dl-5-ethyl-5-phenyloxazolidinedione; PB, phenobarbital; S-9, postmitochondrial subfraction.
I
of active site concentration-response relationships for this type of induction (5). Previous in vivo studies have shown that variation in the alkyl substitution at C-5 of the barbituric acid nucleus (7-12), or in the structure of the heterocyclic ring in a series of ethyl/phenyl-substitutedPB congeners (13,14), is accompaniedby altered ability to induce P450 2B activity in the rat. However, these studies have invariably related inducing ability to the dose of compound administered. Similarly,while dose-responsivenessin P450 2B induction by barbiturate analogues has been demonstrated in previous studies (e.g., refs 8, 9, 15, and 161,no attempts were made in these experiments to relate the degree of induction observed to plasma or hepatic xenobiotic concentration or to any other approximation of the concentration of the xenobiotic at the putative receptor site within the hepatocyte. In the present experiments, the measurement of serum total xenobiotic concentration has been used as a method of approximating steady-state hepatocellular xenobiotic concentration in rats administered inducing agent in the diet for 14 days. The rationale for this is the fact that the hepatocellular plasma membrane contiguous with the
This article not subject to U.S.Copyright. Published 1993 by the American Chemical Society
Pharmacodynamics of P450 2B Induction
5-ethyl-5-phenylbarbituric acid (PB)
for the determination of structure-activity relationships for induction of this subfamily of P450 is demonstrated by the excellent agreement between the relative potencies for these three congeners determined in vivo and in cultured rat hepatocytes.
Experimental Section
H
0
Chem. Res. Toxicol., Vol. 6, No. 2, 1993 189
CHZCH3
5-ethyi-5-phenyl. hydantoin (EPH) H
5-ethyi-5-phenyl. oxazolidinedione (EPO)
Figure 1. Structural formulas of the congeners examined. In the case of EPH and EPO, which have a chiral center at C-5, racemic mixtures were employed.
venous sinusoid, as well as the endothelial lining of the sinusoid itself, are relatively porous, allowing most drug molecules, regardless of physicochemical nature (lipophilicity, ionization),to perfuse the hepatic parenchyma freely (17). P450 2B induction was measured by means of the relatively specific catalytic activities, pentoxy- and (benzy1oxy)resorufin O-dealkylation and testosterone 168hydroxylation, which have been shown to be mediated preferentially by isozymes of the P450 2B subfamily (16, 18-20). By relating the observed inducing effects to the total (free plus protein-bound) xenobiotic concentrations in serum, direct examination of the in vivo potency and efficacy of P450 2B induction in a series of PB congeners should be possible. Doseresponse experiments conducted in cultured adult rat hepatocytes also have the potential to yield data on the pharmacodynamics of P450 induction, and such an approach has recently been employed by Kocarek et al. (21). In a study of the effects of drugs on cellular mRNAs coding for P450 2B1, P450 2B2, P450 3A1, and P450 3A2, these investigators were able to estimate the ED50 values for PB, clotrimazole, and trans-nonachlor. In the present study, we have examined the induction of P450 2B in hepatocyte cultures by measuring increases in (benzyloxy)resorufin O-dealkylation activity. By means of these in vivo and cell culture methodologies, we have examined three ethyllphenyl-substituted congeners which were identified in preliminary studies (14,221 to represent, on an equimolar basis, very effective inducers [PB and 5-ethyl-Bphenylhydantoin(EPH)]or a very weak inducer [5-ethyl-5-phenyloxazolidinedione (EPO)I. The identification of a single substitution within the heterocycle, the replacement of the amido NH group of EPH with an oxygen to yield EPO (Figure 11, is shown to result in an order of magnitude decrease in potency for P450 2B induction.2 In addition, the utility of the culture model These results were presented in part at the 31st Annual Meeting of the Society of Toxicology held in Seattle, Washington, on February 2327, 1992 [Toxicologist 12,401 (Abstract 1582)l.
Chemicals. PB and testosterone were purchased from Sigma Chemical Co. (St.Louis, MO), and 7-pentoxy- and 7-(benzyloxy)resorufin were obtained from Molecular Probes, Inc. (Eugene, OR). Dicumarol and resorufin were purchased from Aldrich Chemical Co. (Milwaukee, WI), and fluorescamine was from Roche Diagnostics (Nutley, NJ). EPH and EPO were synthesized as racemic mixtures as described previously (22, 14). Animal Treatment and Preparation of Samples. Male F344/NCr rats were obtained at 6 weeks of age from the Animal Production Area, Frederick Cancer Research and Development Center (Frederick, MD), and were randomized by body weight at 8 weeks of age into groups of three rata each. The rats were maintained on hardwood chips at 68-72 O F and -50% humidity in an American Association for Laboratory Animal Care-certified laboratory. The various groups received either control diet (Purina Lab Chow No. 5010) or diet containing one of the three test agents at varying concentrations (EPH: 16.3,48.9,147,440, or 1320 ppm; EPO: 442,1326,3978,11934,or 15 000 ppm; PB: 6.17, 18.5, 55.6, 167, 500, or 1500 ppm). After 14 days on test diet, individual body weights were recorded, and the rats were killed by COz asphyxiation. Blood samples were obtained by cardiac puncture from individual rats for the determination of serum levels of the congeners, and livers were removed in toto, trimmed free of extraneous tissue, and weighed. The hepatic tissues were rinsed repeatedly in 0.15 M potassium chloride/0.2 M sucrose (4 OC) until the rinse fluids were devoid of blood and then homogenized in -3 mL/g wet liver weight of the same solution with a Polytron tissue homogenizer (Brinkmann Instruments,Westbury, NY). Postmitochondrial supernatants (S9), resulting from sequential 2000g and 9OOOg centrifugations, were aliquoted into '/*-dram glass vials and stored at -70 OC prior to use in enzyme assays. Microsomal pellets were obtained following centrifugation of individual S-9s (105000g for 75 min), and microsomes for use in testosterone hydroxylase assays were resuspended in 0.05 M phosphate buffer (pH 7.5) containing 0.15 M potassium chloride/0.2 M sucrose. Microsomes to be employed in immunochemical analyses were resuspended in 0.5 M Tris (pH 7.5). Protein content in the S-9 and microsomal samples was determined using fluorescamine (23)or the method of Bradford (24),with bovine serum albumin as the standard. Xenobiotic Analysis in Serum Samples. Prior to analysis, serum samples (300pL) were extracted with 4 mL of ethyl acetate, shaken for 10 min, and then centrifuged (10 min, 2500 rpm). The ethyl acetate layer was transferred to a second vessel. A second extraction with ethyl acetate was performed, and the ethyl acetate layers were combined and evaporated to dryness under nitrogen. The extracts were reconstituted in 300 pL of methanol and subjected to reversed-phase HPLC analysis. For the analysis of EPH or PB, a Cle column (25 cm X 4.6 mm, 5 pm, Burdick and Jackson, Muskegon, MI) was eluted under isocratic conditions with 25 % acetonitrile/75% dibasicsodium phosphate buffer (0.01 M, pH 8.0, EPH analysis) or 20% acetonitrile/80% phosphate buffer (PB analysis) at a flow rate of 1 mL/min. In either case, detection of the analytes was by absorbance at 240 nm. EPO was detected a t 240 nm using an ion-pairing system consisting of an Asahipak ODP-50, 15 cm X 4.6 mm column (Advanced Separation TechnologiesInc., Whippany, NJ) eluted isocratically with 25 % acetonitrile/75% hexyltriethylammonium phosphate (50 pM, pH 10.0,Alltech Associates Inc., Deerfield, IL) at a flow rate of 1.0 mL/min. For all three analytes, the integrated absorbance peak areas were compared to standards prepared by the addition of known amounts of analyte to rat serum. Standards were extracted in parallel with the test sera.
190 Chem. Res. Toxicol., Vol. 6,No. 2, 1993 Preparation of Cultured Rat Hepatocytes. Hepatocytes were isolated from male Fischer rats (220-250 g body weight) as described previously (25). The cell yield was approximately 6 x los hepatocytes per liver, with a viability of 180% ,as determined by trypan blue exclusion. Approximately 5 X lo6cells, suspended in Williams E medium containing M dexamethasone and M insulin, were inoculated on 60-mm plates coated with Matrigel. The plates were incubated a t 37 OC in a humidified atmosphere containing 5% COa, and media were changed daily. After 48 h in culture, the cells were treated for 22 h with dimethyl sulfoxidealone or increasing concentrations of PB, EPH, or EPO, each dissolved in dimethyl sulfoxide. The final concentration of dimethyl sulfoxide in the culture medium was 2 pL/mL. Alkoxyresorufin O-Dealkylase Assays. The O-dealkylation of pentoxy- and (benzy1oxy)resorufinby hepatic S-9 samples was measured with a modification (26) of assays developed originally by Burke et al. (18) and Lubet et al. (16). The final concentration used for each substrate was 5 pM, the S-9 concentrations used ranged from 0.25 to 1.3 mg of protein/mL of reaction mixture, and reaction rates were determined at -25 OC using a Perkin-Elmer LS-50 spectrophotofluorimeter (excitation h = 522 nm, emission h = 586 nm). Increases in relative fluorescence per unit time were compared to the fluorescence of known amounts of resorufin. (Benzy1oxy)resorufinO-dealkylation activity was measured in sonicates prepared from cultured hepatocytes as described previously (25). Testosterone Hydroxylase Assay. The incubations consisted of a 3-mL mixture containin; 50 mM phosphate buffer (pH 7.41, 4 mM NADPH, 250 pM testosterone, 20 pM 11hydroxytestosterone, and 10 pM 17@-(N,N-diethylcarbamoyl)4-methyl-4-aza-5a-androstan-3-one. The latter chemical was added to the reaction mixtures to inhibit steroid 5a-reductase activity (27). The reaction mixtures were preincubated, while shaking, for 5 min at 30 "C and then initiated by the addition of microsomal protein (0.9-2.1 mg). One-milliliter aliquots were withdrawn from each incubation mixture at 2-4 min after the start of the reaction and pipetted directly into 6 mL of dichloromethane, followed immediately by mixing for 30 s on a vortex mixer. The extraction of each reaction mixture was repeated using an additional 6 mL of dichloromethane, and the pooled extracts were evaporated to dryness under a stream of nitrogen. The dried residues were resuspended in a final volume of 300 pL of solvent. A 50-pL aliquot was injected for HPLC analysis using the conditions described by Sonderfan et al. (19). The hydroxylated metabolites of interest were identified and quantified by comparison with authentic standards (Steraloids, Inc., Wilton, NH). Immunochemical Detection of Hepatic P450 2B 1Protein. Immunoreactive CYP2Bl protein in liver microsomal samples was determined by slot-blot analyses, as described previously (14),with a mouse monoclonal antibody (B50) which detects P450 2B1 but does not cross-react with P450 2B2 (28). Analysis of Data. Group mean liver/body weight ratios as well as pentoxy-and (benzy1oxy)resorufin O-dealkylation and testosterone 16@-hydroxylation values were transformed to percentage of maximal response by calculating for each xenobiotic concentration the absolute increase over the control (no xenobiotic) value, and then expressing the increases obtained as a percentage of the maximum increases obtained in the series of concentrations for a given congener. These values were then plotted versus nominal xenobiotic concentration administered or mean serum total xenobiotic concentration, and the resulting data were fitted to the sigmoid E,,, equation (29):
E = E,,,CN/(EC50N
+ CN)
with curve-fitting software (Tablecurve,Jandel Scientific, Corte Madera, CA). In this equation, E is in units of percentage of maximal effect, E,,, is the maximal increase observed for the congener under consideration, Cis the concentration of xenobiotic in diet, serum, or medium, ECm represents the congener concentration associated with a half-maximal response, and N is a curve shape factor. In cases in which complete dietary or
Nims et al. serum concentration-response curves were obtained, the EDso and ECso values were calculated, along with the SE, by the computer program. In cases where the response curves were truncated (missing the top plateau region), the EDsoand ECso values are expressed as "La given concentration". Conformational Analysis. The geometries of the three congeners were optimized with molecular orbital calculation software as described by Nims et al. (14). For purposes of comparison of the three structures, the energy-minimized conformations were superimposed in such a manner as to allow direct overlap of the ethyliphenyl substituents and carbon-5 of the heterocycle.
Results In Vivo Dose-Response Studies with EPH, EPO, and PB. Dietary concentrations for the three congeners were chosen on the basis of preliminary examination of a single dose level (equimolar to 500 ppm PB; 14). An attempt was made to include both no-effect as well as maximal induction effect concentrations. In the case of PB and EPH, no gross toxic effects were observed at the dietary concentrations examined, based upon the absence of decreases in final body weight or body weight gained over the treatment interval (data not shown). However, the rats fed the highest concentrations of EPO (11934 and 15 000 ppm) displayed significantly decreased final body weights compared to the controls (241 f 10 and 218 f 16 g vs 269 f 12 g for the control group, P < 0.05, MannWhitney U-test) and gained less body weight over the 14-day feeding period (25 f 4 and 0 f 7 g vs 42 f 0 g for the control group, P < 0.05, Mann-Whitney U-test). Each of the three congeners caused profound and dietary concentration-dependent induction of hepatic P450 2Bmediated catalytic activities [(benzyloxy)- and pentoxyresorufin O-dealkylation and testosterone 16j3-hydroxylation, Tables 1-1111 and immunoreactive hepatic P450 2B1 protein (Figure 2, Tables 1-111). In general, the ED50 values based upon the four different P450 2B-based end points were similar for any given xenobiotic. However, the concentration-response curves for liver/body weight ratio (increases with which profound P450 2B-type induction is often associated) were shifted to the right compared to the P450 2B-based end points for each xenobiotic. This shift is reflected in the greater EDmvalues for this parameter (Tables 1-111). The three congeners differed with respect to the dietary concentrations required to elicit profound P450 2B induction responses. While equimolar concentrations of PB and EPH appeared to cause approximately the same degree of response, much higher dietary concentrations of EPO were required to elicit any given level of P450 2B induction. This shift to the right in the dietary concentration-induction response curve for EPO is reflected in 20- to 30-fold greater ED50 values for the various end poinls for this congener, compared to PB and EPH (Tables 1-111). The maximal induction responses (Emm) obtained for EPO were 5075 5% of the maximal responses resulting from EPH or PB, the latter congeners causing roughly equivalent responses for the various end points. In Vivo Pharmacodynamics of P450 2B Induction by EPH, EPO, and PB. Steady-state serum total (free plus protein-bound) concentrations for each of the three congeners displayed a linear concordance with nominal dietary congener concentration (Figure3). When the levels of P450 2B-mediated catalytic activity or immunoreactive P450 2B1 protein or liverlbody weight ratio values were
Chem. Res. Toxicol., Vol. 6, No. 2, 1993 191
Pharmacodynamics of P450 2B Induction
Table I. Hepatic Response to Varying Dietary Concentrations of Phenobarbital in the Male F344/NCr Rat dietary phenobarbital concentration (ppm)"
0 6.17 18.5 55.6 167 500 1500 ED50 (ppm) EC50 (PM)
alkoxyresorufin 0-dealkylase benzyloxy pentoxy 24 f 5b 28 f 2 (1.2) 65 f 33d (2.7) 345 f 34d (14) 745 f 126d(31) 1264 f 24d (53) 1289 f 63d (54) 136 f 17 9.8 f 0.1
11 f 2 6 13 f 1 (1.2) 31 f lod (2.8) 112 f 8d (IO) 373 f 46d (34) 661 f 6gd (60) 641 f 50d (58) 144 f 13 10 f 0.2
testosterone 160-hydroxylase
immunoreactive P450 2B protein
liver/ body weight ratio
31 f lb 54 f 18d(1.7) 177 f 66d (5.7) 1176 f 162d (38) 2445 f 393d (79) 2894 f 916d (93) 3661 f 710d (118)
45' 87 (1.9) 166 (3.7) 641 (14) 1107 (25) 1179 (26) 1093 (24)
4.54 f 0.15b 4.60 f 0.42 (1.01) 4.55 f 0.13 (1.00) 4.68 f 0.20 (1.03) 4.74 f 0.17 (1.04) 5.72 f 0.04d (1.26) 6.09 0.08d (1.34)
104 f 21 8.8 f 0.2
53 f 2 6.9 f 0.4
*
336 f 33 27 f 1.7
a Rats were exposed to phenobarbital or control diet for 14 days. Values shown are mean f SD for three F344/NCr rata per treatment (in parentheses, valuetrested/valuecontrol). Units for alkoxyresorufin 0-dealkylation activities are (pmol of resorufin formed/min)/mg of S-9 protein at N 25 OC; units for testosterone 160-hydroxylation activity are (pmol of hydroxylated product formed/min)/mg of microsomal protein at 37 "C; units for liver/body weight ratio are g of liver/100 g body weight. Values are for microsomes pooled from three F344/NCr rats per treatment, in relative densitometric area units per pg of loaded protein (in parentheses, valuetreated/valuecontlo~). Significantly greater than value for rats receiving control diet, P < 0.05, Mann-Whitney U-test.
Table 11. Hepatic Response to Varying Dietary Concentrations of 5-Ethyl-5-phenylhydantoin(EPH) in the Male F344/NCr Rat dietary EPH concentration (ppm)"
alkoxyresorufin 0-dealkylase benzyloxy pentoxy
testosterone 160-hydroxylase
immunoreactive P450 2B protein
0 16.3 48.9 147 440 1320
43 f l l b 60 f 6d (1.4) 337 f 80d (7.8) 901 f 71d (21) 1241 f 187d(29) 1351 A 143d (31)
18 f 4b 27 f 3d (1.5) 149 f 9d (8.3) 447 f 4gd (25) 630 f 81d (35) 836 f 132d (46)
4 f 46 78 f 5d (197) 540 f 92d (135) 1468 f 126d (367) 2216 f 196d (554) 2141 f Illd (535)
114c 174 (1.5) 824 (7.2) 2070 (18) 2151 (19) 2682 (24)
PB
1197 f 12gd (29)
722 f 113d (40)
2241 f 263d (560)
2853 (25)
ED50 (ppm) EC50 (PM)
*
102 5 6.3 f 0.5
171 f 40 7.9 f 0.2
97 f 9 5.9 f 0.8
liver/ body weight ratio 4.28 f O.2Ob 4.43 f 0.10 (1.04) 4.43 f 0.16 (1.04) 4.67 f 0.06d (1.09) 5.04 f 0.16d (1.18) 5.61 f O.lOd (1.31) 5.72 f 0.52d (1.34)
81 f 3 5.5 f 0.4
2353 11f2
a Rats were exposed to EPH or control diet for 14 days. A separate group of rats received P B (500 ppm in the diet). This group served as a concurrent positive control. Values shown are mean f SD for three F344/NCr rats per treatment (in parentheses, valuetreeted/valuecontral).
-
Units for alkoxyresorufin 0-dealkylation activities are (pmol of resorufin formed/min)/mg of S-9 protein at 25 OC; units for testosterone 160-hydroxylation activity are (pmol of hydroxylated product formed/min)/mg of microsomal protein a t 37 OC; units for liver/body weight ratio are g of liveril00 g body weight. Values are for microsomes pooled from three F344/NCr rats per treatment, in relative densitometric Significantly greater than value for rats receiving control diet, area units per pg of loaded protein (in parentheses, valuetreeted/valuecontrol). P < 0.05, Mann-Whitney U-test.
Table 111. Hepatic Response to Varying Dietary Concentrations of 5-Ethyl-5-phenyloxazolidinedione (EPO) in the Male F344/NCr Rat dietary EPO concentration (ppm)"
0 442 1326 3978 11934 15000
alkoxsresorufin 0-dealkvlase benzyloxy pentoxy 43 A 4b 41 f 2 (1.0) 86 f 20d (2.0) 858 f 127d(20) 1078 f 87d (25) 916 f 188d(21)
PB
1472 f 109' (34)
ED50 (ppm) EC50 (PM)
2888 3 124 f 0.0
*
15 f 2b 14 f 1 (1.0) 32 f 7' (2.1) 427 52' (28) 556 f 61' (37) 549 f 74' (37)
*
893 f 118' (60) 3016 f 2 129 f 0.0
testosterone 160-hydroxylase NDc ND 77 f 32b 1087 f 79 1007 f 19 894 f 114 2644 f 1017 21500 276
immunoreactive P450 2B protein 74d 95 (1.3) 173 (2.3) 674 (9.1) 987 (13) 557 (7.5) 1167 (16) 3191 f 71 139 f 3
liver/ body weight ratio 4.06 f 0.25b 4.01 f 0.34 (1.00) 4.12 f 0.17 (1.01) 4.41 f 0.08' (1.09) 4.90 O.Oge (1.21) 4.42 f 0.03' (1.09)
*
5.21 i 0.37d (1.28) 14600 1184
a Rats were exposed to EPO or control diet for 14 days. A separate group of rata received P B (500 ppm in the diet). This group served aa a concurrent positive control. Values shown are mean f SD for three F344/NCr rats per treatment (in parentheses, valuet,eated/valuecontrol).
Units for alkoxyresorufin 0-dealkylation activities are (pmol of resorufin formed/min)/mg of S-9 protein a t -25 OC; units for testosterone 16(3-hydroxylation activity are (pmol of hydroxylated product formed/min)/mg of microsomal protein at 37 OC; units for liver/body weight ratio are g of liver/100 g body weight. ND, product formation was below the limit of detection. Values are for microsomes pooled from three e Significantly F344/NCr rata per treatment, in relative densitometric area units per pg of loaded protein (in parentheses, valuetreated/valuecontro~). greater than value for rats receiving control diet, P < 0.05, Mann-Whitney U-test.
related to serum total congener concentration, the resulting serum concentration-induction response curves displayed the sigmoid shape characteristic of a receptor-mediated effect (plots for the catalytic activities are shown in Figure 4). As observed for the ED50 values, the EC50 values based upon the four different P450 2B-based end points were quite similar for any given congener (Tables 1-111). The concentration-response curves for liverlbody weight ratio again displayed a shift to the right compared to the P450
2B-based end points for each congener, and this was reflected in 1.5- to 3.0-fold greater EC5o values for this parameter (Tables 1-111). The three congeners differed with respect to the EC50 values for P450 2B induction, in that the values for EPO were 10-to 20-fold greater for the various end points, compared to PB and EPH (Tables 1-111). The latter two congeners were approximately equivalent in potency for P450 2B induction, as indicated by the nearly identical EC50 values. Similarly, the serum
192 Chem. Res. Toxicol., Vol. 6, No.2,1993
Nims et al.
PB
1.0
0.33
EPH 0.11
1.0
0
0
6.1 7
16.3
18.5
48.9
55.6
0.33
EPO 0.11
1.0 0 -
0.33
0.11
-414213I26
147
39
167
440 -
11s
500
1320
15C
1500
PB
-
Figure2. Levels of immunoreactive P450 2B1 protein in male F344/NCr rats exposed for 14days to the indicated dietary concentrations (ppm)of phenobarbital (PB), 5-ethyl-5-phenylhydantoin(EPH), or 5-ethyl-5-phenyloxazolidinedione (EPO). The underlined dietary concentrations are equimolar for the three xenobiotics. PB, a t a concentration of 500 ppm, was used as a positive control with the EPH and EPO experiments. Slots in the three lanes of each panel were loaded with 1.0,0.33, or 0.11 pg of pooled (n= 3 rats/treatment) hepatic microsomal protein, as indicated. The primary antibody employed was B50, a mouse monoclonal which detects rat P450 2B1 but does not cross-react with rat P450 2B2 (28). 100
75 50
100 25
0 1
10
100
1000
100 10
75 50 25
0
1
10
100
1000
10000
Drug dose (ppm in diet)
Figure 3. Relationship between dietary xenobioticconcentration and steady-state serum total (freeplus protein-bound) xenobiotic concentration in male F344/NCr rats exposed to phenobarbital (v),5-ethyl-5-phenylhydantoin (a),or 5-ethyl-5-phenyloxazolidinedione (+). Each point represents the mean value for three rats, and the error bars indicate the SD.
concentrations of PB and EPH yielding 20-fold increases in (benzy1oxy)resodi 0-dealkylationactivity were equivalent (8.1 and 7.4 p M , respectively), while 163 p M EPO was required. DoseResponse Studies for EPH, EPO, and PB in Cultured Rat Hepatocytes. (Benzy1oxy)resodin 0dealkylation activity was measured in sonicatesof cultured adult rat hepatocytes exposed for 22 h to culture medium containing varying nominal concentrations of the three congeners. Each of the congeners caused concentrationdependent increasesin the 0-dealkylation activity (Table IV). In confirmation of the results obtained in the in vivo studies, EPO was found in the hepatocyte culture experiments to be much less potent in inducing this monooxygenase activity, with an E D Mof ~ 108 p M , in comparison with ED50 values of 14.5 and 14.2 p M for PB and EPH, respectively (Table IV). The concentrations of PB and EPH yielding 20-fold increases in this activity were 18.8 and 26.6 pM,respectively, as compared with 110 p M in the case of EPO. In terms of the maximal induction Since no attempt was made to measure the actual congener concentrations in culture media over the course of these experiments, the term ED:,,, is used in place of ECra. The use of the latter term is justified only when the xenobiotic concentration in the medium is demonstrated to remain at the nominal concentration over the entire course of the experiment.
l:m 1
10
100
1000
1
10
100
1000
25
0
Total drug in serum (pM)
Figure Pharmacodynamicsof induction of P450 2B-mediaad (benzy1oxy)resodi (A) and pentoxyresorufii (B)0-dealkylation and testosterone 16p (C) hydroxylation activities in male F344/ NCr rats exposed to varying dietary concentrations of phenobarbital (v),5-ethyl-5-phenylhydantoin (a),or 5-ethyl-5phenyloxazolidinedione (+) for 14 days. The serum xenobiotic concentrations shown represent total drug (free plus protein bound). The maximal responses for each congener were determined from the data in Tables 1-111 as described in the Experimental Section. Each point represents the mean value for three rats, and the fit line was computed with curve fitting software.
responses obtained, EPO was found to be approximately equivalent to the other two congeners.
Discussion
To argue convincingly for the role of a receptor in the induction of isozymes of the P450 2B subfamily by PBtype xenobiotics, it must first be established that the induction is dose-responsive and that there are structural requirements for this effect besides those which simply determine lipophilicity (5). Evidence for the dose-responsivenessof PB-type P450 induction in vivo in the rat has previously been reported (8, 9, 15, 16, 30, 31). In addition, the induction of other manifestationsof the PBtype pleiotropic response (including certain isozymes of
Chem. Res. Toxicol., Vol. 6, No. 2, 1993 193
Pharmacodynamics of P450 2B Induction Table IV. (Benzy1oxy)resorufin 0-Dealkylation Activity Measured i n R a t HeDatocute Sonicates ~~~
nominal xenobiotic concn (LIM) 1.0 3.3 10 33 100 333 1000
(benzy1oxy)resorufin 0-dealkylation activity [(pmol/min)/mg of sonicate]
PBn
EPH
EPO
5.0 f 0.5 (1.7)b 9.5 f 1.0 (3.3) 32.5 f 5.1 (11) 86.1 f 14.3 (30) 97.6 f 12.3 (34) 74.3 (27) NT
3.4 f 0.4 (1.2) 8.2 f 0.7 (2.8) 26.0 f 5.7 (9.0) 64.5f 6.3 (22) 72.9 f 6.4 (25) 74.8 (26) NT
NT' NT 3.2 (1.1) 6.0 (2.1) 47.5 (16) 108.6 (37) 68.0 (23)
ED50 (wM) 14.5 f 0.8
14.2 f 0.5
108 f 44
a Abbreviations: PB, phenobarbital; EPH, 5-ethyl-5-phenylhydantoin; EPO, 5-ethyl-5-phenyloxazolidinedione.The values represent the means for 2 or 3 plates per treatment, and where triplicate values were obtained, the standard deviation is given. Values given with in parentheses represent the ratio of activitytreated/activitycontrols, control activity being (2.9 f 0.6 pmol/min)/mg of sonicate protein. ED50 values were calculated with Tablecurve software and are given along with the standard error. NT, not tested.
UDP-glucuronosyltransferase and glutathione S-transferase, and liveribody weight ratio increase) has also been shown to be dose-responsive in the rat (15,321. However, these studies have related the induction response to the dose of xenobiotic administered, rather than to the amount of xenobiotic at the putative receptor site (the microenvironment within the hepatocyte). Likewise, previously reported in vivo structure-induction activity studies (71 4 ) have involved administration of single-dose levels of xenobiotics and/or have failed to consider the concentration of xenobiotic in the fluid surrounding the hepatocyte. In the strictest sense, the determination of relative potency for an effect (such as P450 2B induction) must be based upon consideration of the complete sigmoid active site concentration-response curves for each congener. The present study was designed to allow the direct comparison of the P450 2B induction potency of three structurally related PB-type inducers (PB, EPH, and EPO). In order to examine the pharmacodynamics of hepatic cytochrome P450 induction in vivo, the assumption was made, in the present study, that steady-state serum concentration of the inducing agent represents the closest approximation of xenobiotic concentration at a putative active site within the hepatocellular parenchyma. A range of dietary concentrations for the three congeners was chosen, based upon preliminary examination of a singledose level (equimolar to 500 ppm PB; 141, to include both no-effect as well as maximal-effect concentrations. The duration of administration, 14 days, was chosen to allow steady-state conditions to be attained. Dietary drug administration may be treated kinetically in a manner similar to that employed for constant-rate infusion or repeated single dosing. Thus, regardless of dose, steadystate serum concentrations of the drugs should be attained within 7 elimination half-lives of administration. For PB, with an elimination half-life of 9-11 h in the rat (7,9,33, 34),steady-state conditions should be achieved within 3-4 days of continuous dietary administration. In support of this, we have observed in separate studies that rats administered PB in the diet for 7 days have serum PB levels similar to those in rats administered PB for 14 days (63 f 12 pM,n = 3, vs 52 f 15 pM,n = 9, respectively). Similarly, EPH has been reported to have an elimination half-life of 7.6 h in the rat (351, and on the basis of this
value, steady-state conditions should be achieved within 3 days of dietary administration. By examining the induction of P450 2B under steadystate xenobiotic concentrations, it becomes possible to compare responsiveness to different congeners without having to correct for differences in rate or extent of metabolism, or differences in the temporal kinetics of the induction response to the various xenobiotics. A preliminary study of the ability of a number of ethyliphenylsubstituted structural congeners of phenobarbital to induce P450 2B activity ( 1 4 ) suggested that EPO was markedly weaker as an inducer than PB, EPH, or the other congeners examined. In the present study, this apparent difference in P450 2B-inducing ability of EPO has been demonstrated to represent a 10-fold decrease in potency, as well as approximately 25% lower efficacy, compared with the close structural analogues, PB and EPH. The latter two xenobiotics have now been shown to be approximately equivalent with regard to both potency as well as efficacyfor this effect in the rat. However, there appear to be species differences in the relative ability of EPH and PB to cause P450 2B induction. For instance, while PB and EPH are of equal potency and efficacy in the F344/NCr rat (this paper), in the B6C3F1 mouse, a dietary concentration of 2000 ppm EPH is required to cause the level of P450 2B induction which results from a dietary concentration of 500 ppm PB.4 Pharmacodynamic studies will have to be performed in order to ascertain whether such differences relate to differences in metabolism of the hydantoin in the two species,or whether these findings represent actual species differences in relative potency at a putative induction receptor site. That there may be species differences in relative potency of P450 2B induction by PB congeners is not surprising, given the differences in responsiveness of female and male F344iNCr rats to P450 2B induction by PB. This female/ male differential in F344/NCr rats has recently been shown to represent a 4- to 7-fold decrease in potency in the female, while the efficacy (maximal induction response) is approximately equivalent in the two sexes (36). These differences in potency could be explained by subtle alterations in the binding site of some putative induction receptor, or by changes in the number of available receptor sites between the different sexes, strains, or species. However, such differences are not proof of the existence of receptor mediation in P450 2B induction. While the present in vivo experiments would appear to provide sufficient data to assess potency and efficacy of P450 2B induction, experiments with cultured adult rat hepatocytes also have the potential to elucidate the structure-activity relationships for this effect. Such experiments have only recently become possible, due to the elucidation of culture conditions which allow the expression of this isozyme subfamily following inclusion of PB-type inducers in the culture medium (25, 37, 38). There are certain advantages to conducting such studies in culture, including the fact that it becomes much easier to control the concentrations of drug to be tested and, perhaps more importantly, interanimal variability can be avoided. The practicality of such an approach has recently been demonstrated by Kocarekandco-workers (21). These investigators were able to determine the ED50 values for induction by PB of RNAs coding for P450 2B1, P450,2B2, R. W. Nims, unpublished results.
194 Chem. Res. Toxicol., Vol. 6, No. 2,1993
P450 3A1, and P450 3A2 in hepatocyte cultures derived from adult Sprague-Dawleyrats. The EDwvalues for P450 2B1 and P450 2B2 induction obtained by Kocarek (15 and 5.7 p M ,respectively) are very similar to the ED50 value for induction by PB of (benzy1oxy)resorufin 0-dealkylation activity in hepatocyte cultures determined in the present study (14.5 pM). Importantly, the in vivo ECw values obtained for PB, ranging from 5.6 to 10pM for the various end points (thispaper and ref 36),are essentially equivalent to these hepatocyte culture results. That the cultured hepatocyte results are quite indicative of the results obtained in vivo is also demonstrated in the present study by the agreement between the two systems in the assessment of the relative potency of PB, EPH, and EPO. The in vivo results, taken together with both the results of the present hepatocyte experiments and the published data of Kocarek and co-workers(21)in cultured rat hepatocytes, would appear to substantiate the applicability and relevance of the hepatocyte system directly for the study of P450 2B induction. The examination of EC50 and ED50 values and the use of the sigmoid E, equation for the analysis of the pharmacodynamic data are predicated upon the assumption that receptor occupancy determines the induction response obtained. In receptor theory, the EC50 represents the kdismiation for the xenobiotic-receptor complex, and the maximal response occurs when all available receptors are occupied by ligand (39). Since it is not known whether a receptor is involved in P450 2B induction, it is necessary to consider alternative approaches for the analysis of pharmacodynamic data. One such approach involves the determination of the concentration of xenobiotic required to elicit a certain level of response (40). This approach does not take account of the maximal response elicited by an individual drug and, therefore, is not subject to the errors which would arise if the maximal induction levels were determined by factors not relating to the induction process itself (e.g., toxicity, limits of solubility,etc.). When the in vivo pharmacodynamic data for induction of (benzy1oxy)resorufii0-dealkylationactivitywere analyzed by the latter approach, the results were similar to those obtained with the classicalreceptor-based analysis. Thus, the serum concentrationsof PB and EPH required to cause a 20-fold increase in the 0-dealkylation activity were equivalent (-8 pM), while 163 p M EPO was required for this levelof induction. The correspondingvalues obtained in cultured hepatocytes were 18.8and 26.7 p M for P B and EPH, respectively, as compared to 110 p M for EPO. There are at least two mechanisms by which the rather limited structural difference between EPO and EPH or PB could lead to the marked decrease in potency of EPO, relative to EPH and PB, which was observed for induction of P450 2B in the rat liver or in cultured rat hepatocytes. The first is that induction of P450 2B involves binding of the inducer to a cellular receptor and that EPO lacks a necessary functional group on the heterocycle which the other two congeners possess, the lack of which weakens the interaction of EPO with the receptor. When the minimized molecular structures of the three congeners are superimposed in such a manner that the ethyl and phenyl substituents are aligned, then it is observed that only carbon-5 and the two vicinal atoms (with their respective functional groups) within the heterocycles are in close approximation (Figure 5). This is due not only to the slight deviation from planarity of the heterocycle
Nims et al.
Figure 5. Superimposed energy-minimizedmolecular conformations of phenobarbital, 5-ethyl-5-phenylhydantoin, and 5ethyl-5-phenyloxazolidinedione. The three structures were aligned to allow overlap at carbon-5 of the heterocycle and the ethyl and phenyl substituents. Only one enantiomer for the two racemic compounds (i.e., the hydantoin and oxazolidinedione)is displayed. Color scheme: white, carbon or hydrogen; red, oxygen; blue, nitrogen.
of PB, compared to the planar heterocycles of EPO and EPH, but also to the fact that PB has a six-membered heterocycle while EPH and EPO have five-membered heterocycles (Figure 1). The presence of the ester oxygen in EPO instead of the carbonyl group (PB) or amido NH group (EPH) at one of these two vicinal positions may cause lowered affinity for some binding site and may therefore be responsible for the decrease in potency of the oxazolidinedione. For the purpose of clarity, only one enantiomer each of EPO and EPH have been displayed in Figure 5, despite the use of racemic mixtures in the present experiments. However, due to the symmetry about carbon-5 of the heterocyclein the case of PB, the alignment
Pharmacodynamics of P450 2B Induction of EPH and EPO displayed in Figure 5 is maintained, relative to PB, regardlessof whether the I- or d-enantiomers of EPO or EPH are considered. Alternatively, there is a second possible explanation for the diminished potency of EPO, relative to P B or EPH, which does not require the involvement of a receptor site. In this second mechanism, the decrease in potency could be postulated to arise from the fact that the oxazolidinedione has a markedly lower pKa (-5.5) than the corresponding barbiturate (-7.4) or hydantoin (-8.5) (14). The pka of EPO is sufficiently low, in fact, that at the pH of the blood or intracellular fluid (-7.4), EPO exists primarily in the ionized form (14). While this most likely would not impede the congener from perfusing into the hepatocyte, for reasons given above, the fact that the xenobiotic is in the ionized form might be expected to inhibit diffusion of the congener into lipid membranes. It willbe of interest, therefore, to examine the inducing ability of an analogue of EPO in which the acidic proton (the proton of the amido NH group) is replaced, for instance, with a trifluoromethyl or tert-butyl group. Such a structural analogue of EPO would not contain an ionizable proton and, therefore, would be expected to have greater potency than the parent compound if the nonspecific (nonreceptor-mediated) mechanism applies. However, this analogue might still lack potency if the receptor-mediated mechanism applies. In summary, we have now demonstrated active site concentration/in vivo P450 2B induction response relationships for PB and two structural congeners and have shown that there are differences in potency and efficacy among the three xenobiotics. Similar pharmacodynamic studies for in vivo P450 induction have not been undertaken previously by other investigators. While Pei and co-workers investigated the relationship between steadystate serum PB concentration and antipyrine clearance in the rat (33),the authors were not able to demonstrate a linear concordance between the two parameters. The in vivo pharmacodynamics for the anticonvulsant action of PB have been studied in the rat (34), and the EC50for this effect,based upon total serum PB concentration, was found to be 327 pM. As in the case of P450 2B induction by PB, the mechanism for anticonvulsant action by PB has yet to be determined. However, it is of interest that the potency of PB for anticonvulsant action is 30- to 50-fold lower than the potency for P450 2B induction. The results of the present study provide evidence that structure-P450 2B induction relationships in cultured adult rat hepatocytes closely approximate the in vivo structure-activity relationships for this effect.
Acknowledgment. We are grateful to H. Walton, D. Logsdon, C. Driver, G. Macgruder, L. Riffle, and J. Carter for excellent technical assistance. This project has been funded at least in part with Federal funds from the Department of Health and Human Services under Contract N01-CO-74102 with Program Resources, Inc. The content of this publication does not necessarily reflect the views or policies of the Department of Health and Human Services, nor does mention of trade names, commercial products, or organizations imply endorsement by the US. Government.
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