Comparative induction of CYP3A and CYP2B in rat liver by 3-benzoylpyridine and metyrapone

Chemico-Biological Interactions 113 (1998) 161–173

Comparative induction of CYP3A and CYP2B inrat liver by 3-benzoylpyridine and metyrapone

Michael Murray *, Rachel M. Sefton, Robert Martini,Alison M. Butler

Storr Li6er Unit, Department of Medicine, Uni6ersity of Sydney, Westmead Hospital, Westmead,NSW 2145, Australia

Received 6 October 1997; received in revised form 29 January 1998; accepted 30 January 1998

Abstract

3-Benzoylpyridine (3BP) is a major metabolite of HGG-12, an oxime that has beensynthesized as a potential antidote to the toxic effects of soman and other anti-cholinesterases. Structural similarities exist between 3BP, the cytochrome P450 (CYP)-in-ducer metyrapone (MET) and other 3-substituted pyridines that interact with CYPs. Thepresent study evaluated the regulatory effects of 3BP on CYP expression in rat liver. Both3BP and MET (100 mg/kg) increased total hepatic microsomal holo-CYP content signifi-cantly 24 h after administration to male rats. Pronounced increases in activities mediated byCYP2B (androstenedione 16b-hydroxylation and 7-pentylresorufin O-depentylation) wereproduced by 3BP and MET, which correlated with respective 9- and 14-fold increases inCYP2B immunoreactive protein. In addition, both agents slightly increased rates of microso-mal CYP3A-dependent steroid 6b-hydroxylation, troleandomycin metabolite complex for-mation and total CYP3A immunoreactive protein. Induction of the dexamethasone-inducibleCYP3A23 mRNA to 4.5- and 2.5-fold of control was detected in liver of MET- and3BP-induced rats; CYP3A2 mRNA levels were unchanged. Analogous in vitro studiesrevealed that MET was a preferential inhibitor of CYP3A-mediated steroid 6b-hydroxyla-tion activity, but 3BP was inactive against constitutive steroid hydroxylase CYPs. Thesefindings indicate that the structurally related 3BP and MET elicit similar induction effects onCYPs 2B and 3A23 in rat liver after in vivo administration, but differential inhibitory effects

* Corresponding author. School of Physiology and Pharmacology, University of New South Wales,Sydney, NSW 2052, Australia. Tel.: +61 2 93852561.

0009-2797/98/$19.00 © 1998 Elsevier Science Ireland Ltd. All rights reserved.

PII S0009-2797(98)00017-9

M. Murray et al. / Chemico-Biological Interactions 113 (1998) 161–173162

of the chemicals on CYP activity in vitro. Recent reports have implicated a microsomalbinding site in the induction of CYP3A1/3A23 in rat liver. In light of the present findings,substituted pyridines like 3BP may be useful tools in structure–activity studies to evaluatethe physicochemical requirements for binding to this protein. © 1998 Elsevier Science IrelandLtd. All rights reserved.

Keywords: Cytochrome P450; Gene regulation; 3-Benzoylpyridine; Metyrapone

1. Introduction

The pyridine heteroaromatic system is common to many chemicals of toxicolog-ical significance. The 3-substituted pyridine NNK (4-[methylnitrosamino]-1-[3-pyridyl]-1-butanone) is formed by the nitrosation of nicotine during tobacco curingand is activated by cytochromes P450 (CYPs) to DNA-alkylating metabolites [1,2].A number of pyridine-based molecules are used as therapeutic agents, including theantihistamines, pyrilamine and tripelennamine, the antibacterial drug, sulfapyridine,and the anticholinergic agent, tropicamide.

Pyridine congeners induce CYPs soon after administration to mammals. Pyridineitself upregulates CYPs 2B, 2E1 and 4B in rabbit liver [3] and CYPs 1A1/2, 2B1/2and 2E1 in the rat [4,5]. Substituted pyridines, especially those containing alkyl- oraryl- moieties at the 3- or 4-positions of the heteroaromatic ring, are also inducersof these CYPs [6–9]. Metyrapone (1,2-[dipyrid-3%-yl]-2-methyl-propan-1-one; MET)is a 3-substituted pyridine that has been used previously for the determination ofpituitary function by inhibiting adrenal steroidogenesis and upregulates CYPs 1A,2B and 3A after in vivo administration to rats [10–12].

The oxime HGG-12 (3-benzoylpyridino[1]methyl-2%-hydroxyiminomethylpyridino[1%]methyl ether dichloride) has been developed as a potential antidote to the toxiceffects of soman and other anticholinesterases [13]. HGG-12 is metabolized rapidlyto 3-benzoylpyridine (3BP), which has also been detected in aqueous solutions ofthe oxime shortly after preparation. 3BP and MET are structurally similar.

Certain 3-substituted pyridines are also inhibitors of hepatic microsomal CYPactivity in vitro. Because of its clinical value in the inhibition of steroidogenesis,MET is probably the most studied agent, but there is evidence that structurallysimilar pyridines also bind to and inhibit CYP activities [7,14]. In the present studythe capacity of 3BP, a product of HGG-12 biotransformation and decomposition,to influence the expression and function of CYPs was investigated in rats. Theprincipal finding to emerge was that in rat liver 3BP, like MET, upregulated themicrosomal content and activity of the phenobarbital-inducible CYP2B and dexam-ethasone-inducible CYP3A23, a gene that is homologous to CYP3A1. (CYP3A23and CYP3A1 differ by only 26 or 29 bases, the former has a six-base deletion andboth genes appear to be regulated similarly [15–17].)

In in vitro studies MET was an effective inhibitor of CYP3A-dependent steroid6b-hydroxylation, but 3BP was inactive against major constitutive CYPs in rat

M. Murray et al. / Chemico-Biological Interactions 113 (1998) 161–173 163

liver. Thus, 3BP and MET satisfy structural requirements for the induction ofCYP2B and 3A23 in vivo, although their in vitro interactions with CYPs are quitedifferent.

2. Materials and methods

2.1. Chemicals

[4-14C]Androst-4-ene-3,17-dione (androstenedione; specific activity, 56–59 mCi/mmol) and [a-32P]dCTP (specific activity, 3000 Ci/mmol) were purchased fromAmersham Australia (North Ryde, NSW, Australia). Androstenedione metabolitestandards were obtained from Steraloids (Wilton, NH), Sigma Chemical Co. (St.Louis, MO) or the MRC Steroid Reference Collection (Queen Mary’s College,London, UK). 3BP, MET, DBD [2,3-(dipyrid-3%-yl)-butane-2,3-diol], 7-pentylre-sorufin (resorufin 7-O-pentyl ether), troleandomycin (TAO) and biochemicals forenzyme assay were from Sigma. Solvents (HPLC grade) and chemicals (analyticalreagent grade) were obtained from Rhone-Poulenc and Ajax (Sydney, Australia),respectively. CYP-specific oligonucleotides were synthesized by Bresatec (Adelaide,South Australia) and the 18S RNA oligonucleotide was obtained from Pac-Bio PtyLtd (Rushcutters Bay, NSW, Australia). Oligonucleotide sequences were: CYP3A2,5%-ACT-GCC-TTT-GTG-AAG-ATC-CCA-ATA-AAA-TTC-3% (reverse comple-ment of nucleotides 1594–1623 [17]); CYP3A23, 5%-CTT-TCA-CAG-GGA-CAG-GTT-TGC-CTT-TCT-CTT-GCC-3% (reverse complement of nucleotides 482-514[15]); and 18S RNA, 5%-CGG-CAT-GTA-TTA-GCT-CTA-GAA-TTA-CCA-CAG-3% [18].

2.2. Animals and preparation of hepatic microsomal fractions

Protocols were approved by the Western Sydney Area Health Service AnimalEthics Committee. Male Wistar rats (�250 g) were from the Animal CareDepartment, Westmead Hospital, and were housed in cages under conditions ofconstant temperature and lighting (12-h day/night cycle).

Animals received a single dose of one of the test compounds (100 mg/kg i.p. incorn oil) or three doses of phenobarbital or dexamethasone on consecutive days [19]and were killed 24 h later. Livers were removed, perfused with cold saline and snapfrozen for storage at −70°C, until required for the isolation of microsomalfractions or RNA. Washed microsomal pellets were suspended in potassiumphosphate buffer (50 mM, pH 7.4) that also contained 20% glycerol and 1 mMEDTA, and were stored at −70°C until required [20]. Microsomal protein andtotal holo-P450 were estimated by the procedures of Lowry et al. [21] and Omuraand Sato [22], respectively. In some experiments, holo-P450 content was determinedafter the incubation (20 min) of TAO (250 mM) and NADPH (1 mM) withmicrosomal fractions (1 mg protein/ml) from variously pretreated rats.


2.3. Assays of microsomal androstenedione hydroxylation

[14C]Androstenedione (50 mM; 0.18 mCi/0.4 ml incubation) hydroxylation assays(0.15 mg microsomal protein; 37°C) were conducted in potassium phosphate buffer(0.1 M, pH 7.4; 1 mM EDTA) [23]. Reactions were started with NADPH (1 mM)and were terminated 2.5 min later by the addition of chloroform (5 ml) and transferto an ice bath. Steroid metabolites were extracted into the organic phase which wasevaporated to dryness and the residue was applied to TLC plates (Merck silica gel60 F254 type; Darmstadt, Germany). Plates were developed twice in chloroform/ethyl acetate (2:1), with air drying between [24]. Radioactive metabolites comigratedwith authentic standards and were detected by autoradiography (Hyperfilm-MP;Amersham) before quantification by scintillation counting (ACS II; Amersham).

In some experiments, varying concentrations of 3BP and MET as potential CYPinhibitors were added to reaction tubes in acetone. The solvent was removed undera stream of nitrogen and steroid hydroxylation reactions were then conducted asdescribed.

2.4. Assay of microsomal 7-pentylresorufin O-depentylation

The microsomal O-dealkylation of 7-pentylresorufin was monitored by thecontinuous spectrofluorometric assay of Prough et al. [25]. Reactions (2 ml),contained 0.2 mg microsomal protein and the substrate concentration was 10 mM.

2.5. Detection of CYPs in rat li6er microsomes by immunoblotting

Microsomal fractions were heated for 5 min at 100°C with 2% sodium dodecylsulfate and 5% b-mercaptoethanol, and loaded onto vertical polyacrylamide gels(7.5%; 20 lanes per side). For CYP3A blots, 5 mg of protein (control liver, 10 mg;dexamethasone-induced, 2 mg) were loaded onto each lane. For CYP2B blots, 30mg of microsomal protein from untreated and DBD-treated rats were loaded, 5 mgfrom 3BP- and MET-treated rat liver and 1 mg from phenobarbital-treated rat liver;these quantities were in the linear response range. Gels were electrophoresedovernight at �15 mA per side, and then proteins were transferred electrophoreti-cally to nitrocellulose sheets (Schleicher and Schuell, Dassel, Germany). Nitrocellu-lose sheets were then washed sequentially in the solutions described previously [26].

The primary antibodies that were raised in rabbits and that were available forthese studies (dilutions used in immunoblotting are indicated in parenthesis) werean anti-rat CYP3A IgG (3.7 mg protein/ml; supplied by Dr A. Astrom, KarolinskaInstitute, Stockholm) and an anti-rat CYP2B1 IgG that had been cross-adsorbedagainst CYP2B1 that had been immobilized on CNBr-activated Sepharose 4B (0.08mg protein/ml; [27]). CYP immunoreactive proteins were detected on Hyperfilm-MP(Amersham) by enhanced chemiluminescence and autoradiography. The resultantsignals were analyzed by laser densitometry (Molecular Dynamics Personal Densit-ometer SI, Sunnyvale, CA, USA).


2.6. Extraction of total RNA from rat li6er and analysis of CYP mRNAs

Total RNA was extracted from male rat liver by the guanidinium thiocyanate/CsCl method [28]. For mRNA analysis of CYP3A2, CYP3A23 and 18S oligonucle-otides were labelled using [a-32P]ATP and deoxynucleotidyl transferase. RNA (10mg) was electrophoresed on 1% agarose in the presence of 2.2 M formaldehyde andthen transferred to Hybond-N+ nylon filters (0.45 mm, Amersham). Hybridizationand washing conditions were as described previously [23], and signals correspond-ing to CYP mRNAs were quantified using a Molecular Dynamics Phosphorimager.To demonstrate equivalence of RNA loading between samples, filters were strippedand rehybridized to an a-32P-labeled probe to 18S ribosomal RNA.

2.7. Statistics

Data are presented throughout as mean9S.E.M. Significant differences betweenmeans of two or three groups were detected, respectively, by Student’s t-test orDunnett’s q %-test following one-way analysis of variance.

3. Results

3.1. Effects of 3BP and MET on microsomal CYP-mediated substrate oxidation in6i6o.

The content of total holo-P450 determined spectrophotometrically in hepaticmicrosomes from untreated rats was 0.8090.01 nmol/mg protein, and was in-creased �25% after administration of either 3BP or MET 24 h before sacrifice(1.0090.05 and 1.0090.08 nmol/mg protein, respectively; Table 1; PB0.05). Incontrast, the structural analogue DBD (Fig. 1), had no effect on total microsomal

Table 1Effect of in vivo administration of pyridines on cytochrome P450 and microsomal androstenedioneoxidation in rat liver

Treatment holo-P450 Hydroxyandrostenedione metabolite (nmol/mg protein/min)(nmol/mg

16a-protein) 16b- 6b- 7a-

0.4590.033.7590.270.4290.03None (control) 2.2690.180.8090.014.3990.27 0.6090.02*MET (100 mg/ 1.0090.08** 4.4490.17* 1.9090.25**

kg)2.7290.200.8090.04 0.4890.02DBD (100 mg/ 3.2790.180.4490.04

kg)0.6990.05*4.6490.402.3590.60*4.8390.79*1.0090.05**3BP (100 mg/kg)

Different from control: *PB0.01, **PB0.05.


Fig. 1. Structures of chemical used in this study: 3BP, DBD and MET.

holo-P450 content (0.8090.04 nmol/mg protein). Further studies evaluated theparticular CYPs that were upregulated after administration of 3BP and MET.From Table 1 it emerges that certain pathways of androstenedione hydroxylationwere increased in rat liver after administration of 3BP and MET. The most notableincrease was in the activity of the 16b-hydroxylation pathway which is catalyzed byCYP2B1; 3BP and MET produced increases to 5.6- and 4.5-fold of control activity(Table 1). The activity of the 16a-hydroxylation pathway (CYPs 2C11/2B1 [24])was increased to about 2-fold of control (PB0.01) with smaller increases in steroid7a-hydroxylation activity (CYPs 2A1/2 [29]). The increase in 16a-hydroxylation isconsistent with the apparent induction of CYP2B1. In contrast, a trend towards anincrease in the 6b-hydroxylation pathway was noted, but this did not attainstatistical significance. Again, DBD did not influence any pathway of androstene-dione hydroxylation in rat liver (Table 1).

From a consideration of the observed rates of steroid hydroxylation, it appearedthat CYP2B proteins may be upregulated by 3BP and MET, but not DBD. Tofurther evaluate this possibility, rates of CYP2B-mediated 7-pentylresorufin O-de-pentylation were measured in hepatic microsomes from 3BP- and MET-inducedrats. Consistent with previous reports, this activity was extremely low in untreatedrat liver and was approximately 17- and 21-fold greater following administration of3BP and MET to male rats (Fig. 2A; PB0.01). By comparison, phenobarbital, the

Fig. 2. Effects of 3-substituted pyridines on (A) 7-pentylresorufin O-depentylation and (B) TAO-MIcomplexation in rat liver microsomes. Different from control: *PB0.01, **PB0.05.


Fig. 3. Western immunoblots of CYP2B (upper) and CYP3A (lower) proteins in hepatic microsomesfrom untreated (CONT), MET- or 3BP-pretreated rat liver (signals in microsomal fractions from twoindividual livers per treatment are shown). Immunoblots for CYP2B in microsomes from phenobarbital(PB)-induced rat liver and CYP3A in dexamethasone (DEX)-induced rat liver are also indicated forreference. Protein loadings were: CYP2B (CTL, 30 mg; 3BP and MET, 5 mg; and PB, 1 mg), CYP3A(CTL, 10 mg; 3BP and MET, 5 mg; and DEX, 2 mg). The appearance of at least two proteinsimmunoreactive with the anti-CYP3A IgG is typical of our Wistar rat strain.

prototypic inducer of CYPs 2B, increased 7-pentylresorufin oxidation about 250-fold (not shown).

Although CYP3A-dependent steroid 6b-hydroxylation activity was not signifi-cantly influenced by the administration of either 3BP or MET to male rats, therehave been reports that MET is an inducer of specific CYP3A subfamily proteins inrodents [11,12]. The macrolide antibiotic TAO is converted by inducible CYP3Aproteins to a reactive metabolite that forms an inhibitory complex with CYPs 3A[30]. Thus, sequestration of holo-P450 as a TAO metabolite intermediate-complexwas determined in hepatic microsomes from untreated and 3BP-, MET- anddexamethasone-pretreated male rats. From Fig. 2B it is apparent that, in micro-somes from untreated rat liver, only 0.0590.01 nmol P450/mg protein (�6% oftotal) formed a complex with the TAO metabolite. In contrast, in microsomes from3BP- and MET-induced rat liver �13 and �11% of total P450 (0.1490.04 and0.1390.03 nmol P450/mg protein, respectively) was available for complexation. Asanticipated, a large proportion of P450 (�44% of total) generated a TAOmetabolite complex in microsomes from dexamethasone-treated rats (0.5190.03nmol/mg protein). Thus, the possibility emerged that 3BP and MET may modulatethe relative expression of 3A subfamily CYPs in liver.

3.2. Effects of 3BP and MET administration on CYP apoprotein and mRNAexpression

Evidence was obtained from catalytic studies that CYP2B proteins were inducibleby 3BP, as well as by MET. Immunoblotting experiments confirmed the upregula-tion of CYP2B protein by 3BP and MET (to �9- and �14-fold of control,respectively), but not DBD, and that phenobarbital produced an approximateincrease of CYP2B-immunoreactive protein to about 27-fold of control (Fig. 3).Note that different amounts of microsomal protein from control and induced rat


liver were loaded onto gel lanes and this is probably responsible for the weakersignal for the faster migrating CYP2B protein in microsomes from MET-, 3BP- andPB-induced rat liver. Total CYP3A immunoreactive protein was increased by about40% by both 3BP and MET. As anticipated, dexamethasone was a potent inducerof total CYP3A protein (Fig. 3).

Because CYP3A proteins are catalytically similar and are cross-reactive withcommonly used IgG preparations, the effect of 3BP and MET administration torats on major CYPs 3A was investigated further at the mRNA level. Fig. 4 presentsa series of Northern blots that indicate upregulation of hepatic CYP3A23 mRNAafter treatment of male rats with 3BP (to 250920% of control; PB0.001) andMET (to 4509100% of control; PB0.02). It is also evident that CYP3A2 mRNAwas unaffected by administration of these agents to rats. These findings in relationto the relative inducibilities of CYPs 3A2 and 3A23 by dexamethasone areconsistent with the recent report of Mahnke et al. [31]. In the same study it was alsodemonstrated that the mRNAs for two other CYPs 3A, namely CYP3A9 andCYP3A18, are present in untreated rat liver at extremely low levels. The presentfindings establish that, like DEX, MET and 3BP exert differential effects on theexpression of CYPs 3A2 and 3A23 in rat liver.

3.3. In 6itro inhibition of constituti6e CYP microsomal oxidation acti6ities by 3BPand MET

From the foregoing experiments it was clear that 3BP and MET exerted similareffects on CYP 2B and 3A gene expression in vivo. MET is also a well establishedin vitro inhibitor of CYP reactions, and therefore interacts with CYPs by multiplemechanisms. As part of the present study the in vitro effects of 3BP on majorconstitutive CYPs was assessed in rat liver (Fig. 5). MET was a potent inhibitor of

Fig. 4. Northern analysis for CYP3A23 and CYP3A23 mRNAs in liver from untreated (CONT) andMET- or 3BP-pretreated male rats. The 18S signal was used to correct for differences in RNA loadingprior to quantification.


Fig. 5. Inhibitory effects of 3BP (�) and MET () on microsomal androstenedione 2a-, 6b-, 7a-, 16b-and 16a-hydroxylations in vitro. Data are estimates from duplicate incubations that varied by less than8% from the indicated means.

CYP3A-mediated androstenedione 6b-hydroxylation (IC50=18 mM at an an-drostenedione concentration of 50 mM) and also modulated the activities of the16a-, 16b- and 7a-hydroxylation pathways to a lesser extent. 3BP did not signifi-cantly affect any of the four principal pathways of androstenedione hydroxylationthat are mediated by constitutively expressed CYPs.

4. Discussion

The present study describes the potency of 3BP, a 3-substituted pyridine that isgenerated in situ during the enzymic and nonenzymic hydrolysis of thecholinesterase reactivator HGG-12, as an effective inducer of CYPs 2B and 3A23 inrat liver. The regulatory properties of 3BP were quite similar to those exhibited byMET, another 3-substituted pyridine that has been well studied for its inhibitoryand inductive effects on CYPs. Thus, it is likely that the structural requirements forinduction of CYPs 2B and 3A23 are satisfied by both 3BP and MET. In contrast,


3BP and MET produced different effects on microsomal CYP activities in vitro.MET was a preferential inhibitor of CYP3A activity, but 3BP did not inhibit anypathway of steroid hydroxylation in untreated rat liver.

Several recent studies have evaluated the regulatory effects of pyridines on CYPexpression. Pyridine rapidly induces CYPs at the levels of gene transcription ormRNA stabilization (CYPs 1A [4] and 2B [5]) or by enhancing the rate of mRNAtranslation (CYP2E1 [32]). Studies of CYP2B induction by substituted pyridineshave demonstrated that the nature of ring substitution influences the potency andprofile of CYP induction. Thus, marked induction of CYPs 2B was noted 24 h afteradministration of 4-benzoyl- and 4-a-hydroxybenzylpyridine [8]. However, effectsof pyridines on other inducible CYPs were not evaluated.

Understanding of the mechanisms of induction of some CYPs by chemicals isincreasing [33]. For example, potent inducers of CYP1A have been shown to bindand activate the Ah receptor that initiates gene transcription. The factors con-trolling CYP2B induction are not as well understood. Some studies have implicatedDNA-response elements that accommodate barbiturate-inducible proteins (re-viewed in Ref. [33]). CYP3A enzymes are strongly induced by chemicals such asTAO and phenobarbital, but upregulation by dexamethasone, pregnenolone 16a-carbonitrile and related steroids led to the association of inducible CYPs 3A withthe glucocorticoid receptor [33]. However, the relative responsiveness of CYPs 3Aand tyrosine aminotransferase to glucocorticoids were quite different. Thus, doubtwas cast over the involvement of a glucocorticoid receptor in CYP3A induction.Recently a hepatic microsomal ‘receptor’ that can accommodate a number ofstructurally distinct inducers of CYP3A has been documented [34]. This site bindsdexamethasone with an affinity about 25-fold lower than that of the glucocorticoidreceptor; MET also binds to this site. There have been additional studies that haveimplicated specific nucleotide sequences in the 5%-flanking regions that mediatetranscription of the CYP3A1 [35] and CYP3A23 [36] genes. From the findings inthe present paper it is feasible that 3BP and MET may also interact with the siteresponsible for CYP3A23 transcriptional activation. CYPs 3A are also upregulatedby inhibitors of 3-hydroxy-3-methylglutaryl-CoA reductase (HMG-CoA reductase),such as lovastatin [37] and the bisphosphonate SR-12813 [38]. Thus, it has beensuggested that an association may exist between the control of cholesterol biosyn-thesis and CYP3A gene regulation.

If CYP3A23 upregulation is mediated in part by interaction of 3BP and METwith a microsomal binding site, the hydrophobic nature of these agents does notappear to be a major determinant of induction. Log10 P values for 3BP, MET andDBD were similar (calculated using tabulated values and the additivity principle[39]) to be 1.70, 1.89 and 1.96, respectively. Clearly, DBD was inactive as a CYPinducer, even though it was similarly hydrophobic to MET and 3BP. Differencesbetween these calculated values are small and are unlikely to have a significantbearing on either the pharmacodynamics of the analogues in vivo or hydrophobicinteractions with cellular macromolecules involved in gene transcription.

Several classes of nitrogen heterocycles, including pyridines, are effective CYPinhibitors [14,40]. In the present study both 3BP and MET interacted with oxidized


CYP in untreated rat liver to elicit type II optical difference spectra (not shown).Interactions of this type are commonly produced by nitrogen heterocycles andreflect the interaction of the nitrogen lone-pair of electrons with the CYP heme [41].However, 3BP did not significantly inhibit hepatic microsomal CYP activity, so thatits capacity to remain bound throughout the CYP reaction cycle (involving sub-strate binding, CYP reduction and oxygen activation) appears low. Despite thesepoints, 3BP is reportedly an inhibitor of thromboxane synthetase, which hasprompted some interest in the potential of such agents as anticoagulants [42]. It isnoteworthy that the catalytic cycle of thromboxane synthetase, an atypical CYP,does not involve the usual process of electron transfer from NADPH via theflavoprotein reductase [43].

In conclusion, the present findings indicate that administration of 3BP and METin vivo leads to the induction of CYP2B and CYP3A23 expression in rat liver. Both3-substituted pyridines bind to oxidized CYP heme in the absence of CYP sub-strates, but only MET remains bound to subsequent intermediates in the CYPreaction cycle and elicits enzyme inhibition in vitro. Several classes of CYPinhibitors have been shown to induce those CYPs to which they bind and inhibit[41]. In contrast, 3BP is a chemical that induces CYP3A but is an ineffectiveinhibitor of CYP3A activity. From such considerations, pyridine analogues of thetype described may prove useful in further characterizing the microsomal CYP3Areceptor binding site or trans-acting factors that bind to the 5%-flank and activateCYP3A transcription.

Acknowledgements

The generous gift of anti-CYP3A IgG by Dr Anders Astrom, KarolinskaInstitutet, Stockholm, (present address: Astra Draco Co., Lund), Sweden is grate-fully acknowledged. This work was supported by a grant from the AustralianNational Health and Medical Research Council.

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Comparative induction of CYP3A and CYP2B in rat liver by 3-benzoylpyridine and metyrapone

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