Benzo[e]pyrene elicits changes in the biochemical activities and chromatographic behavior of murine hepatic cytochromes P-450 that are distinct from those induced by...

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Chem.-Biol. Interactions, 83 (1992) 203-220 203 Elsevier Scientific Publishers Ireland Ltd.

BENZO[e]PYRENE ELICITS CHANGES IN THE BIOCHEMICAL ACTIVITIES AND CHROMATOGRAPHIC BEHAVIOR OF MURINE HEPATIC CYTOCHROMES P-450 THAT ARE DISTINCT FROM THOSE INDUCED BY 2,3,7,8-TETRACHLORODIBENZO-p-DIOXIN

JACQUELINE A. GIBBONS and JOHN G. BABISH

Department of Pharmacology, New York State College of Veterinary Medicine, Cornell University, Ithaca, New York 14853 (USA)

(Received October 22nd, 1991) (Revision received April 14th, 1992) (Accepted April 14th, 1992)

SUMMARY

The objective of this study was to examine the potential for a specific ligand of carcinogen binding protein (CBP) to induce changes in the overall character of hepatic microsomal cytochromes P-450 (P450) and to compare potential changes with those induced by an Ah receptor ligand. Benzo[e]pyrene (BeP) was previously shown to bind CBP with high affinity and Ah receptor with low aft- nity. In contrast, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) binds Ah receptor avidly and CBP weakly. Hepatic microsomes were prepared from C57BL/6J (B6) and DBA/2J (D2) mice treated with corn oil, BeP or TCDD. Relative to corn oil controls, pretreatment of B6 mice with BeP or TCDD increased the nmol P450/mg microsomal protein content 26 and 28%, respectively. In D2 mice, nmol P450/mg microsomal protein was increased 23% in the BeP pretreatment, while TCDD pretreatment had no effect relative to the corn oil controls. For the O- alkyl ethers of resorufin, rates of metabolism (per nmol P450) were affected dif- ferently in B6 and D2 by BeP pretreatment. Pentoxyresorufin O-dealkylase ac- tivity was reduced to 44% of control activity in B6 mice and increased 39%

Correspondence to: J.A. Gibbons, Chiron Corporation, 4560 Horton St., Emeryville, CA 94608, USA. Abbreviations: A415, absorbance at 415 nm; AHH, aryl hydrocarbon hydroxylase; B6, C57BL/6J; P450, cytochromes P-450; BeP, benzo[e]pyrene; CBP, carcinogen binding protein; D2, DBA/2J; DMSO, dimethyl sulfoxide; ELISA, enzyme linked immunosorbant assay; HPLC, high performance liquid chromatography; ip, intraperitoneal; MAb, monoclonal antibody; MOR, EOR, POR, BOR, methoxy-, ethoxy-, pentoxy- and benzyloxyresorufin; MROD, EROD, PROD, BROD, methoxy-, ethoxy-, pentoxy- and benzyloxyresorufin O-dealkylation; PAH, polycyclic aromatic hydrocarbon; PBS, phosphate-buffered saline; NADPH, nicotinamide adenine dinucleotide phosphate; TCDD, 2,3,7,8-tetrachlorodibenzo-p-dioxin; Tris, tris(hydroxymethyl)aminomethane; UDP, uridine 5- diphosphate.

0009-2797/92/$05.00 © 1992 Elsevier Scientific Publishers Ireland Ltd. Printed and Published in Ireland

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relative to controls in D2 mice. BeP pretreatment had no effect on ethoxy- resorufin O-dealkylase activity in B6 mice, while this activity was decreased to 58% of controls in D2 mice. Additionally, benzyloxyresorufin O-dealkylase activ- ity was reduced to 65% of control levels in B6 mice and not affected in D2 mice. Methoxyresorufin O-dealkylase activity was reduced in both strains to an average of 55% of control values. As expected, TCDD pretreatment resulted in increases of all O-dealkylations measured in both strains of mouse. For both in- bred strains of mouse, anion exchange chromatography revealed a P450 peak associated with BeP pretreatment that was not present in chromatograms generated with corn oil or TCDD pretreatments. Results of enzyme linked im- munosorbant assays also indicated that the pattern of P450 isoenzyme expres- sion associated with BeP pretreatment was distinct from that associated with TCDD pretreatment. Overall, these data show that treatment with a specific ligand of CBP induces changes the biochemical activities and chromatographic behavior of P450 isozymes in murine hepatic microsomes. Moreover, they in- dicate that changes in P450 occurring after treatment with a CBP ligand are distinct from those changes that are associated with treatment with an Ah recep- tor ligand (TCDD). Differences between B6 and D2 strains suggest that the hepatic P450 changes occurring in response to pretreatment with a CBP ligand may be influenced by the presence of Ah receptor.

Key words: 4S Carcinogen binding protein - Ah receptor -- Enzyme induction -- Benzo[e]pyrene -- 2,3,7,8-tetrachlorodibenzo-p-dioxin - Cytochromes P-450

INTRODUCTION

Exposure to xenobiotics is often associated with an adaptive increase in metabolic biotransformation. Administration of certain chemicals to animals has been shown to result in increased activities of P450 (reviewed in Ref. 1), glutathione transferases [2], epoxide hydrolases [3] and UDP- glucuronyltransferases [4]. These observations have prompted investigators to examine the possibility that xenobiotics exert their induction effect by interac- ting with receptor proteins that are present in target tissues. To date, only one such receptor protein, the Ah receptor, has been identified. Ah receptor has been shown to bind TCDD and its structural congeners with high affinity [5] and to subsequently enhance the production of a distinct set of biotransforming en- zymes (e.g. IA family of P450) [6]. However, for most xenobiotics and families of P450, evidence for an induction mechanism that involves a receptor protein is currently lacking. Thus, it is presently unclear whether the mode of action of Ah receptor may be used to characterize the general mechanism by which xenobiotics enhance the expression of biotransforming enzymes.

In addressing this issue, we have focused on CBP, an intracellular protein that binds pyrene and its derivatives with high affinity. CBP is similar to Ah receptor

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in several important respects: (i) both interact with high affinity to a limited number of xenobiotic ligands [7-11]; (ii) both are markedly hydrophobic [11 - 13]; (iii) both are detectable in xenobiotic metabolizing tissues such as those derived from liver, lung and kidney in most mammalian species [12,14,15]; (iv) both interact with compounds such as benz[a]anthracene, benzo[a]pyrene, dibenz[a,h]anthracene, 3-methylcholanthrene, 5,6- and 7,8-benzoflavone rever- sibly and with high affinity; and (v) neither interacts specifically with compounds such as dexamethasone, cholesterol, phenobarbital, progesterone, estradiol, cor- tisol, or dihydrotestosterone [7- 9]. The extent of similarity between these two proteins has led some investigators to suggest that the 4S CBP is merely a subunit of the 9S Ah receptor [1]. This contention seems unlikely considering the studies of Safe and coworkers [7,8], which have demonstrated the existence of ligands that distinguish one protein from the other. BeP and TCDD represent such ligands. BeP has been shown to bind CBP with high affinity and Ah recep- ter with low affinity; conversely, TCDD binds CBP with low affinity and Ah receptor with high affinity.

Noting the similarities between CBP and Ah receptor, we have proposed that CBP may also mediate gene expression. This hypothesis seems reasonable in light of recent findings by Arnold and coworkers [16]. These investigators demonstrated that following administration of 3-methylcholanthrene to rats, CBP translocates from the cytosol into the nucleus.

The present study was designed to examine the potential for a specific ligand of CBP to induce changes in the overall character of hepatic microsomal P450. Our approach was to characterize P450-mediated activities and to assess physical changes in the pool of isozymes through immunologic and chromatographic techniques. The results indicate that the overall character of P450 in B6 and D2 mice is influenced by pretreatment with BeP, a ligand having a high affinity for CBP. In addition, we show that the pattern of change in the character of P450 associated with a CBP ligand is distinct from the changes associated with an Ah receptor ligand (TCDD).

MATERIALS AND METHODS

Chemicals and animals All chemicals were purchased from commercial sources and were of the

highest purity available. B6 and D2 mice were obtained from Harlan Sprague-Dawley (Indianapolis, IN) and Charles River Laboratories (Wilm- ington, MA), respectively. MAb-containing mouse ascites fluids were provided by Dr. S.S. Park (NCI/NIH, Bethesda, MD). Experiments were performed using 4-6-week-old female mice. Mice were fed Prolab RMH 1000 rat, mouse and hamster food (Agway, Cortland, NY) and received tap water ad libitum. All mice were housed three to five per cage and maintained on a photoperiod of 12 h.

Pretreatment of mice Mice were killed 24 h following an i.p. injection of BeP (50 mg/kg), TCDD (2

~g/kg), or corn oil alone; the volume of the injections ranged from 0.1 to 0.2 ml

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per mouse. Preparation and - 80 ° storage of the microsomes were performed as described [17].

P450 determinations P450 per mg microsomal protein concentrations were determined using the

technique of Omura and Sato [18]. The nmol P450 was calculated from the car- bon monoxide difference spectrum of dithionite-reduced microsomes using a molar extinction coefficient of 91 cm -1 m1-1 for the difference in absorbance between 420 and 490 nm [19].

O-dealkylation assay of ethers of resorufin Resorufin (hydroxyphenoxazone) and the O-alkyl ethers of resorufin, MOR,

EOR, POR and BOR, were purchased from Molecular Probes (Junction City, OR). EROD, MROD, PROD and BROD were measured fluorimetrically as previously described [20,21]. Recordings were standardized by measuring the fluorescence of freshly generated resorufin from resorufin acetate (Sigma, St. Louis, MO) prior to each assay. The concentration of microsomal protein was ap- proximately 0.1 mg/ml for all assays. Reactions were run at 37 ° and were in- itiated by the addition of NADPH. In all cases, rates of metabolism are reported as nmol resorufin formed/nmol P450/min.

In vitro inhibition of O-dealkylation by BeP To assess the in vitro effect of BeP residues on MROD activity, 0.2 mg

microsomal protein and BeP, ranging from 0 to 25 ng (added in 2.5~1 DMSO), were added to 2 ml total volume of reaction mixture. Reaction mixture consisted of 5 ~M MOR, 1 mM NADP, 5 mM isocitrate, 5 mM MgC12 and 50 mM potassium phosphate (pH 7.8). The reaction was initiated by adding 0.1 unit of isocitrate dehydrogenase/ml reaction mixture. MROD was measured as previously described. Inhibition was reported as the rate of reaction with addi- tion of BeP relative to the rate when DMSO alone was added. Preincubation of the microsomes with BeP (rather than combining BeP with the microsomes im- mediately before measuring MROD) was shown not to influence the resultant ex- tent of inhibition.

Hepatic microsomal BeP residues B6 and D2 mice were given i.p. injections (0.1-0.2 ml) of 0, 25, 50, or 100 mg

BeP (in corn oil)/kg body weight and killed 24 h later. Microsomes were prepared as previously described and 5 mg of microsomal protein from each animal was extracted twice with 5 ml octane. The octane was evaporated under nitrogen gas and the residue resuspended in 2 ml octane. The efficiency of BeP extraction by this method was greater than 95°7o at 0.5, 2.5, and 10 ng BeP/mg protein. The concentration of BeP in the final 2 ml of octane was determined fluorimetrically (excitation: 329 nm, emission, 357 nm) [22]. In order to correct for matrix effects, standard curves of BeP fluorescence were generated from direct addi- tion of BeP to octane extracts of hepatic microsomes from corn oil pretreated animals. The relationship between fluorescence and BeP concentration was linear for BeP concentrations ranging from 1 to 40 ng/ml octane extract.

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ELISA ELISA were performed with 96-well microtiter plates precoated with antigen.

Solubilized microsomes were used as antigen and were prepared as a series of geometric dilutions ranging from 1 to 500 fmol P450/ml PBS. One-tenth milliliter from each dilution of P450 was promptly pipetted into wells of a microtiter plate in quadruplicate. Plates were incubated for 2 h at 22 °. After washing with 0.05% Tween-20 (polyoxyethylenesorbitan monolaurate) in PBS, individual wells were saturated with 0.2 ml bovine serum albumin (20 mg/ml in PBS) for 2 h at 37 °. Wells were then incubated with MAb (0.25 × ~g IgG/ml ascites fluid in I mg/ml bovine serum albumin and 0.05% Tween-20 in PBS) and bound for 0.5 - 1.5 h at 22°C. Incubation with horseradish peroxidase-linked an- timouse IgG (Sigma, St. Louis; 0.5 #g IgG/ml PBS) and subsequent washing was performed as with the original MAb. Peroxidase activity was determined after incubation with substrate (0.03% H202 and 1 mM 2,2'-azino-di(d-ethyl- benzthiazoline sulfonic acid-6) dissolved in 100 mM sodium citrate (pH 4.2) at 22 °. At the first visualization of color change, A415 values were recorded. In- dividual wells were measured in the order of substrate addition to control for the effect of time. The dilution containing 8 fmol P450/ml was chosen to compare the concentrations of epitopically-defined isozymes among the pretreatments. This decision was based on the saturation of A415 values at concentrations greater than 8 fmol P450/ml and the low resolution of differences among pretreatments below this concentration. Each experiment consisted of ELISA on each of the three pretreatments (corn oil, BeP and TCDD) within a strain of mouse (B6 or D2). Thus, A415 values were compared only for individual MAb across the pretreatments within a strain of mouse.

HPLC-generated P450 profiles Anion exchange chromatography was performed as previously described [23].

Briefly, 1 mg of solubilized microsomal protein in a volume of 0.1 ml was applied to an Anpac anion exchange column (Anspec, Ann Arbor, MI). Flow rate was 1.0 ml/min and eluting proteins were monitored using an integrator attached to a spectrophotometric detector set at 417 nm. After a 5-min wash to remove non- absorbing proteins, the chromatogram was developed using a 20-rain linear gra- dient of 0 - 400 mM sodium acetate in 20 mM Tris-acetate buffer (pH 7.4), con- taining 0.4% Emulgen 911 (polyoxyethylene alkyl aryl ether, Kao Atlas, Tokyo, Japan) and 20% glycerol. Funae and Imaoka have shown that excellent reproducibility of the tracing at 417 nm is obtained under these conditions [23]. In our experiments, individual trials on the HPLC were discounted if the absorp- tion peaks indicated by the integrator did not conform to those observed for other trials within the pretreatment. One minute fractions were collected and the P450 content of each fraction was determined by the difference spectrum technique as described previously in this report. This procedure was repeated for each of three to five mice in a pretreatment; microsomal protein from one mouse was injected onto the column at a time. Within each of the pretreatments, the fractions in which individual peaks of P450 eluted from the column were highly reproducible.

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Protein determination Bicinchoninic acid was used for the spectrophotometric determination of pro-

tein concentration as described [24].

Statistical analysis Data were analyzed by means of analysis of variance for two factors with un-

equal n among the treatments. The two factors were strain (B6 and D2) and chemical pretreatment (corn oil vehicle, BeP and TCDD). Differences among the treatments were deemed significant when the probability of rejecting the null hypothesis when true was less than 5%. Tukey's lsd was used to determine dif- ferences among means. Confidence limits (95%) were computed using the residual mean square of the analysis of variance and the appropriate n value for the treatment. All statistical calculations were as described [25].

RESULTS

P~50 determinations Concentrations of hepatic P450 per mg microsomal protein were increased in

all pretreated B6 mice relative to the corn oil controls (Table I); both BeP and TCDD were associated with comparable increases of 26 and 28%, respectively. TCDD pretreatment was not associated with a statistically significant increase in hepatic P450 concentrations in D2 mice, while a 23% increase in hepatic microsomal P450 was observed in BeP pretreated D2 mice.

O-dealkylation assay of ethers of resorufin O-dealkylations were used as enzyme markers for the induction of specific

isozymes of P450. EOR is considered reflective of P450IA1 mediated activity,

TABLE I

P450 CONCENTRATIONS IN HEPATIC MICROSOMES FROM VARIOUS PRETREATMENTS OF B6 AND D2 MOUSE STRAINS

Data are given as nmol/mg protein and are the means of determinations of N animals ± S.E.M.

Strain/Pretreatment P450 concentration N

B6 Corn Oil 0.404 ~- 0.058 7 BeP 0.509 ± 0.062* 6 TCDD 0.519 ± 0.050* 7

D~ Corn Oil 0.393 ± 0.032 10 BeP 0.483 ~- 0.069* 7 TCDD 0.425 s: 0.040 6

*P < 0.05 compared to corn oil pretreatment of respective strain.

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while MOR represents P450IA1 and IA2, POR is indicative of P450IIB1 and IIB2 and BOR represents P45011B1 and IIB2 as well as P450IA1 [26-28]. As expected, TCDD pretreatment was associated with increased rates of dealkyla- tion (per nmol P450) of all the resorufin substrates in both B6 and D2 mice relative to the corn oil controls (Table II). These increases were greater overall for B6 than D2 mice. Among substrates, the greatest increases in enzyme activ- ity with TCDD pretreatment were noted for MROD and EROD.

In striking contrast to the findings with TCDD pretreatment in B6 microsomes, BeP pretreatment of this strain was associated with depressed rates of dealkylation (per nmol P450) of all derivatives of resorufin except EOR. In D2 mice, BeP pretreatment resulted in a 40% decrease in P450IA associated activities of MROD and EROD; PROD activity was increased 40% and no effect was noted for BROD.

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Mierosomal BeP residues Residues of BeP likely to be present in the hepatic microsomes of pretreated

mice might interfere with the in vitro assay of resorufin metabolism. To test this possibility, an experiment was performed in which three mice per group were given i.p. injections of 0, 25, 50, or 100 mg BeP/kg and killed after 24 h. Microsomes were prepared as in previous experiments and analyzed to deter- mine BeP residues following in vivo administration. For the 25, 50 and 100 mg BeP/kg doses, hepatic microsomes from B6 mice were determined to contain 7, 38 and 55 ng BeP/mg protein; D2 microsomes contained 15, 29 and 47 ng BeP/mg protein, respectively. Because MROD appeared to be the most sensitive of the resorufin derivatives to BeP pretreatment, MOR was chosen as the model substrate in in vitro BeP inhibition experiments. Rates of MROD in microsomes from corn oil-pretreated mice, to which BeP was added at the time of the assay, were compared to rates of MROD in microsomes from BeP-pretreated animals. These are referred to as in vitro and ex vivo additions of BeP, respectively.

As shown in Fig. 1, MROD was inhibited less than 15% at the highest concen- tration of BeP following in vitro addition for either strain of mouse. For both B6

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and D2 microsomes, the rates of MROD were always lower in microsomes from BeP pretreated animals than in control microsomes to which BeP was added in vitro. Significantly, these experiments indicate that the isozymes that dealkylate MOR constitute a smaller proportion of the metabolically active isozymes in microsomes derived from BeP-pretreated mice than in the controls.

ELISA ELISA were used to compare the levels of P450 apoproteins among the

pretreatments. Note that ELISA were performed with the same concentrations of P450 among pretreatments; differences among pretreatments therefore reflect changes in the proportions of individual isozymes. MAb 1-7-1 has been previously shown to be specific for P450IA1 and IA2 in the mouse, while MAb 1-12-3 binds P450IA1 [28]. TCDD pretreatment increased the proportions of P450 isozymes cross-reactive with MAb 1-7-1 and MAb 1-12-3 in B6 mice (Fig. 2), a finding that is consistent with the well documented effect of TCDD on the enhanced expression of P450IA1 and P450IA2. In D2 mice, MAb 1-7-1 demonstrated an increase in apoprotein with TCDD pretreatment, but no such change was indicated with MAb 1-12-3.

MAb 2-66-3 has been shown to recognize P450IIB1 and IIB2 in the mouse [29]. This antibody had comparable levels of cross-reactivity across all pretreatments, demonstrating that P450IIB1 and IIB2 concentrations were not influenced by either pretreatment. Overall, BeP pretreatment did not significantly affect the hepatic microsomal proportions of P450IA1, P450IA2, P450IIB1 or P450IIB2 apoproteins in either strain of mouse.

HPLC-gene~'ated P450 profiles An HPLC equipped with an anion exchange column was used to separate the

isozymes of P450. By quantitating the P450 in 1 min fractions that eluted from the column, it was possible to obtain 'isozymic profiles' of hepatic P450 in each pretreatment. Isozymes of P450 having similar ionic charges co-eluted from the column. Each peak of P450 therefore consisted of an isoenzyme or set of isozymes that was electrostatically distinct from isozymes eluting in other peaks.

Electrostatic factors that potentially influenced the elution of a particular isoenzyme included ionic charge associated with primary amino acid sequence and secondary modifications such as phosphorylation or glycosylation.

A distinct peak, approximately centered at fraction 19 (Fig. 3) was observed in solubilized hepatic microsomes from BeP-pretreated mice of both strains and an additional peak at fraction 14 was observed in D2 hepatic microsomes. These chromatograms suggested that: (i) BeP pretreatment affected the physical character of isoenzyme(s) in the pool of hepatic microsomal P450; and (ii) the set of isozymes in hepatic microsomes from B6 mice pretreated with BeP had physical characteristics that were distinct from the set associated with the TCDD pretreatment.

DISCUSSION

PAH carcinogens and related compounds have long been believed to exert

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their effects on xenobiotic metabolism by binding either covalently or non- covalently to several cellular constituents. Historically, binding of these com- pounds to components in murine hepatic cytosol has been characterized by sucrose density gradient centrifugation and by gel permeation chromatography. In such studies, investigators have reported that PAH bind chiefly to two cytosolic components: an 8 to 9S (9S) component and a 4 to 5S (4S) component [30,31]. Binding of PAH to the 9S component in murine hepatic cytosols can be reversed by the presence of 100-fold molar excess of TCDD. However, TCDD has no effect on PAH binding to the 4S component. Moreover, the 9S component segregates with the Ah b allele in genetic crosses with mice, but the 4S compon- ent is not associated with the Ah b allele that governs AHH responsiveness [32l. The 9S component, termed Ah receptor, has been well established as a transcrip- tional regulator of several genes; the regulation of the expression of CYPIAI (the P450IA1 gene) by Ah receptor accounts for its role in AHH induction (reviewed

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in Ref. 6). Noting the similarities that exist between the 4S protein (also termed CBP) and Ah receptor, several authors have proposed that these two receptors may serve some of the same functions.

Much work has been done recently to test whether expression of P450IA1, an isoenzyme under the transcriptional regulation of Ah receptor, is also controlled by CBP. Using AHH as a marker of P450IA1 expression, Zytkovicz [33] showed that there was no correlation between the amount of CBP and the inducibility of hepatic microsomal AHH in New Zealand White rabbits by PAH. Harris et al. [34] have reported a similar lack of correlation in both whole animal and cell culture experiments; using + CBP and -CBP Sprague- Dawley rat substrains and + CBP/-CBP rat hepatoma cell lines, they concluded that the presence of CBP did not affect AHH inducibility by Ah or CBP receptor ligands.

In striking contrast to these reports, Houser and coworkers have concluded that CBP plays a direct role in enhancing the expression of P450IA1 [10,35,36]. Genetic reconstruction experiments were performed indicating that CBP com- plexes with specific 5'-upstream regions of CYPIAI (the P450IA1 gene) and stimulates in vitro transcription of a marker gene.

In the present study, P450IA1 expression was further examined. Unlike previous studies, however, this study further addressed the potential for CBP to function in the regulation of P450 isozymes in addition to those of the P450IA gene family. We examined the potential for BeP pretreatment to elicit qualitative and quantitative alterations in hepatic P450 in mice. As a ligand, BeP binds CBP with high affinity and Ah receptor with low affinity [7]. Our experi- mental design included a TCDD pretreatment. TCDD, which has low affinity for CBP and high affinity for Ah receptor [8], provided a basis for comparison of potential CBP and Ah receptor-mediated responses of hepatic microsomal P450. In addition, our design incorporated B6 and D2 mouse strains. These strains ex- press comparable levels of CBP [32], but differ with respect to Ah receptor. D2 mice express a mutant form of Ah receptor, having a depressed capacity to mediate the transcriptional response elicited by Ah receptor ligands [37]. Work by Poland and Glover [38] has shown that higher doses of Ah receptor ligands are required to elicit the same response in D2 mice as that observed in the B6 strain. By comparing BeP-induced changes in these two strains, it is possible to gain insight as to whether actions of CBP ligands may be influenced by the presence of Ah receptor.

The O-alkyl ethers of resorufin, namely MOR, EOR, POR and BOR, were used as diagnostic probes for the induction of distinct isozymes. As made evident by the TCDD pretreatment of B6, the lack of absolute specificity of the hepatic microsomal P450 isozymes for single substrates makes the identification of P450 isozymes by means of individual substrates difficult. In the B6 mice pretreated with TCDD, increased biotransformation of all resorufin derivatives was observ- ed relative to the controls. POR has been reported to be specific for murine P450IIB1 and IIB2 [27]. However, immuno-quantification showed that P450IIB1 and IIB2 apoproteins were not expressed in TCDD pretreated mice at concentrations higher than controls. Apparently, murine P450IA1 and IA2 catalyze the O-dealkylations of MOR, EOR, POR and BOR, yet have the greatest catalytic activity toward MOR and EOR [28].

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Depressed rates of metabolism were observed for the methoxy-alkyl ether of resorufin in both B6 and D2 strains with BeP pretreatment. Considering the fin- dings of our experiments comparing in vitro and ex vivo inhibition by BeP, it is likely that these depressed rates of metabolism reflect changes in the propor- tions of metabolically active P450 isozymes that are present in BeP pretreated mice. Since total hepatic P450 levels are increased in BeP pretreated animals, the ratios of P450 isozymes would differ from those of control and TCDD pretreated mice.

We have addressed the possibility that BeP acts directly as an inhibitor of the enzymes that carry out the dealkylations of the various resorufin derivatives. Notably, our experiments do not address the potential for reactive metabolites of BeP (generated in vivo) to inhibit the metabolism of substrates studied in vitro. In view of previous studies of BeP metabolism [39- 41], however, such a mechanism seems unlikely. In general, the most likely candidates for monooxy- genase inhibition are electrophilic reactants capable of binding target in- tracellular nucleophilic macromolecules such as DNA and proteins. Yet Jacob et al. [42] have shown that such molecular species are not generated in the course of BeP metabolism in rodent hepatic microsomes from uninduced animals, nor after the animals are pretreated with the CBP-specific ligands, BeP and pyrene.

For the BeP pretreatment in both mouse strains, our immunologic data cor- relate poorly with our enzymologic data. For example, based on enzymologic evidence, P450IA2 and IIB [1,2] appear to represent different proportions of the population of isozymes in microsomes derived from BeP pretreated mice than the corn oil controls. ELISA data indicate, however, that these isozymes are in equal proportions in these two pretreatments. This apparent discrepancy can be understood by recognizing that these two types of assay measure different variables; enzymologic assays measure proportions of metabolically active isozymes, yet immunologic assays measure proportions of apoprotein.

That apoprotein proportions do not correspond with the proportions of metabolically active isozymes suggests thatcatalytic activities may be modified by BeP pretreatment. One possible mechanism that might account for such at- tenuation is posttranslational modification of specific isozymes. Mounting evidence in the literature indicates that short-term regulation of P450-dependent monooxygenase activity occurs by phosphorylation. Phosphorylation of P450 has been shown to be reversible [43-45], isoenzyme-selective [44,46-50] and can result in either enhanced [43,51,52] or depressed [48,53-55] rates of P450- mediated catalysis. The intriguing possibility that BeP elicits changes in the phosphorylated condition of isozymes of P450 is worthy of consideration in future studies.

Interestingly, BeP pretreatment was associated with a 56% decrease in biotransformation of POR in B6 mice and a 40% increase in POR metabolism in D2 mice. BROD activity was depressed 35% in B6 mice and not affected in D2 mice following BeP pretreatment. Additionally, anion exchange chromatograms revealed a peak at 14 rain (Fig. 3) in D2 mice pretreated with BeP that was not present in B6 mice similarly pretreated or in D2 controls. The possibility that these differences can be attributed to strain differences in Ah receptor is an in- triguing one (Ah receptor may act coordinately with CBP). However, this

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possibility cannot be adequately addressed in these experiments. Because B6 and D2 are each highly inbred strains which maintain profound genetic differences, they are imperfect models for investigating specific Ah-mediated events. Thus, to investigate more thoroughly the possibility that CBP-mediated events are in- fluenced by Ah receptor, our laboratory has initiated studies of BeP pretreat- ment in Ah congenic mice (substrains which differ only at the chromosomal segment containing the Ah genetic locus).

In common with several previous studies [23,56,57], we have used anion ex- change HPLC to profile changes of P450 subpopulations. Isozymic profiles generated by this technique provide the most dramatic evidence that BeP pretreatment is associated with marked physical alterations in the pool of P450 isozymes. For B6-derived microsomes, the peaks of P450 eluting from the column were different for the corn oil, BeP and TCDD pretreatments; this sug- gests differences in the populations of P450 in each of the pretreatments. These differences are not necessarily a result of increased gene expression. Since the HPLC separation of P450 isozymes is a function of only the ionic charge of the proteins, it is possible that posttranslational modification (phosphorylation, for example) could account for altered retention times. Nonetheless, from these data we are able to conclude that: (i) BeP pretreatment is associated with certain physical changes in the pool of P450 isozymes, relative to corn oil controls; and (ii) BeP pretreatment affects changes in P450 that are distinct from those associated with TCDD pretreatment.

The present study provides no evidence that the effects associated with BeP pretreatment are mediated through CBP, but serves to demonstrate that in vivo exposure to a CBP ligand alters the composite characteristics of murine hepatic P450 isozymes. These changes are clearly distinct from the phenobarbital- and Ah receptor-mediated changes that have been well characterized in the literature. Studies are currently underway in our laboratory to characterize the specific effects that BeP administration may have on the biotransformation of xenobiotics by hepatic microsomal P450. Perhaps most significantly, this study has shown that diagnostic probes focusing only on P450IA1, IA2, IIB1 and IIB2 proteins are likely to overlook major alterations in the distribution profile of hepatic P450 isozymes. In a recent report [58] we demonstrated that BeP pretreatment of B6 mice results in increased bioactivation of aflotoxin B1 in vitro. Future studies of the role of CBP as a direct mediator of the expression of P450 will require a broader spectrum of diagnostic probes.

The results of this study indicated that the biochemical activities and physical character of hepatic P450 in mice is influenced by pretreatment with BeP, a high affinity ligand for CBP. It was also demonstrated that the pattern of alterations in P450 associated with a CBP ligand is distinct from that of an Ah receptor ligand (TCDD). Finally, B6 and D2 strain differences suggest that the hepatic P450 changes occurring in response to pretreatment with a CBP ligand may be influenced by the presence of Ah receptor.

ACKNOWLEDGEMENTS

Dr. Fred Friedman of the Division of Molecular Carcinogenesis, National In-

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stitutes for Health is acknowledged for his helpful review of the manuscript. This work was supported in part by funds from the USDA Hatch Act, administered by the New York State College of Veterinary Medicine, Cornell University, In- terregional Research Project No. 4, USDA/CSRS, 86-CRSR-2-2975 and by the Public Health Service training grant ES-07052 from the Division of En- vironmental Health Sciences, National Institutes of Health. The US Government is authorized to produce and distribute reprints for governmental purposes not- withstanding any copyright notation that may appear hereon.

REFERENCES

1 F.J. Gonzalez, The molecular biology of cytochrome P450s, Pharm. Rev., 40 (1989) 243-288. 2 P. Grolier, P. Cassand, E. Antignac, J.F. Narbonne, R. Albrecht, V. Azais, L.W. Robertson and

F. Oesch, Effects of prototypic PCBs on benzo[a]pyrene mutagenic activity related to vitamin A intake, Mutat. Res., 211 (1989) 139-145.

3 L.V. Kravchenko, E.E. Kuz'mina and V.S. Sobolev, (Comparative characteristics of microsomal epoxide hydrolase in the rat liver and small intestine mucosa (Original)) Sravnitel'naia kharakteristika mikrosomal'noi epoksidgidrolazy pecheni i slizistoi obolochki tonkoi kishki krys, Vopr. Med. Khim., 34 (1988) 122-128.

4 M.H. Grant, S.J. Duthie, A.G. Gray and M.D. Burke, Mixed function oxidase and UDP- glucuronyltransferase activities in the human Hep G2 hepatoma cell line, Biochem. Pharmacol., 37 (1988) 4111-4116.

5 M. Romkes, J. Piskorska-Pliszczynska, B. Keys, S. Safe and T. Fujita, Quantitative structure- activity relationships: analysis of interactions of 2,3,7,8-t tetrachlorodibenzo-p-dioxin and 2- substituted analogues with rat, mouse, guinea pig and hamster cytosolic receptor, Cancer Res., 47 (1987) 5108- 5111.

6 D.W. Nebert and J.E. Jones, Regulation of the mammalian cytochrome P1-450 (CYP1A1) gene, Int. J. Biochem., 21 (1989)243-252.

7 C. Kamps and S. Safe, Binding of polynuclear aromatic hydrocarbons to the rat 4S cytosolic binding protein: structure-activity relationships, Cancer Lett., 34 (1987) 129-137.

8 J. Piskorska-Pliszczynska, B. Keys, S. Safe and M.S. Newman, The cytosolic receptor binding affinities and AHH induction potencies of 29 polynuclear aromatic hydrocarbons, Toxicol. Lett., 34 (1986) 67- 74.

9 N.H. Heintz, B. Tierney, E. Bresnick and W.I. Schaeffer, Correlation of induction of aryl hydro- carbon hydroxylase in cultured rat hepatocytes with saturable high-affinity binding of 3- methylcholanthrene to a 4S cytoplasmic protein, Cancer Res., 41 (1981) 1794-1802.

10 W.H. Houser, R. Zielinski and E. Bresnick, Further characterization of the polycyclic aromatic hydrocarbon binding properties of the 4S protein, Arch. Biochem. Biophys., 251 (1986) 361-368.

11 B. Tieruey, S. Munzer and E. Bresnick, The isolation and characterization of specific 3- methylcholanthrene-binding proteins from rat liver cytosol, Arch. Biochem. Biophys., 225 (1983) 826- 835.

12 T.H. Zytkovicz, Identification and characterization of a high-affinity saturable binding protein for the carcinogen benzo(a)pyrene, Cancer Res., 42 (1982) 4387-4393.

13 M.S. Denison, L.M. Vella and A.B. Okey, Structure and function of the Ah receptor for 2,3,7,8- tetrachlorodibenzo-p-dioxin. Species difference in molecular properties from mouse and rat hepatic cytosols, J. Biol. Chem., 261 (1986) 3987-3995.

14 T.A. Gasiewicz and G. Rucci, Cytosolic receptor for 2,3,7,8-tetrachlorodibenzo-p-dioxin. Evidence for a homologous nature among various mammalian species, Mol. Pharmacol., 26 (1984) 90 - 98.

15 H.A. Barton and M.A. Marietta, Kinetic and immunochemical studies of a receptor-like protein that binds aromatic hydrocarbons, J. Biol. Chem., 263 (1988) 5825-5832.

16 P.S. Arnold, B. Tierney and R.C. Garner, Rat liver immuno-ultrastructural localization of the specific (4-5S) 3-methylcholanthrene-binding protein; evidence for its involvement as a receptor protein in cytochrome P-4501A1 induction, Carcinogenesis, 9 (1988) 21- 28.

Page 16

218

17 C. DeDuve, R. Wattiaux and P. Buadhuim, Distribution of enzymes between subcellular frac- tions in animal tissues, Adv. Enzymol., 24 (1962) 291-358.

18 T. Omura and R. Sato, The carbon monoxide-binding pigment of liver microsomes. I. Evidence for its hemoprotein nature, J. Biol. Chem., 239 (1964) 2370-2378.

19 T. Omura and R. Sato, The carbon monoxide-binding pigment of liver microsomes. II. Solubilization, purification and properties, J. Biol. Chem., 239 (1964) 2379- 2385.

20 M.D. Burke and R.T. Mayer, Ethoxyresorufin: direct fluorimetric assay of a microsomal O- dealkylation which is preferentially inducible by 3-methylcholanthrene, Drug Metab. Dispos., 2 (1974) 583- 588.

21 M.D. Burke, S. Thompson, C.R. Elcombe, J. Halpert, T. Haaparanta and R.T. Mayer, Ethoxy-, pentoxy- and benzyloxyphenoxazones and homologues: a series of substrates to distinguish be- tween different induced cytochromes P-450, Biochem. Pharmacol., 34 (1985) 3337-3345.

22 E. Sawicki, T.R. Hauser and T.W. Stanley, Ultraviolet, visible and fluorescence spectral analysis of polynuclear hydrocarbons, Int. J. Air Poll., 2 (1960) 253-272.

23 Y. Funae and S. Imaoka, Simultaneous purification of multiple forms of rat liver microsomal cytochrome P-450 by high-performance liquid chromatography, Biochim. Biophys. Acta, 842 (1985) 119-132.

24 P.K. Smith, R.I. Krohn, G.T. Hermanson, A.K. Mallia, F.H. Gartner, M.D. Provenzano, E.K. Fujimoto, N.M. C-oeke, B.J. Olson and D.C. Klenk, Measurement of protein using bicinchoninic acid (published erratum appears in Anal. Biochem., 163(1) (1987) 279), Anal. Biochem., 150 (1985) 76-85.

25 G.W. Snedecor and W.G. Cochran, Statistical Methods, 7th edn., The Iowa State University Press, Ames, 1980.

26 H.Y. Yang, M.J. Namkung and M.R. Juchau, Cytochrome P-450-dependent biotransformation of a series of phenoxazone ethers in the rat conceptus during early organogenesis: evidence for multiple P-450 isoenzymes, Mol. Pharmacol., 34 (1988) 67- 73.

27 H. Raunio, A. Kojo, R. Juvonen, P. Honkakoski, P. Jarvinen, M.A. Lang, K. Vahakangas, H.V. Gelboin, S.S. Park and O. Pelkonen, Mouse hepatic cytochrome P-450 isozyme induction by 1,4- bis[2~3,5-dichloropyridyloxy)]benzene, pyrazole and phenobarbital, Biochem. Pharmacol., 37 (1988) 4141 - 4147.

28 J.A. Gibbons, Assignment of ethoxy- and methoxyresorufln O-dealkylase activities to specific isozymes in the murine P450IA protein family by antibody inhibition, in: J.A. Gibbons, Ph.D. Dissertation, 4S Carcinogen Binding Protein as a Potential Mediator of Cytochrome P-450 In- duction, Cornell University, Ithaca, NY, 1991, pp. 163-182.

29 K.C. Cheng, H.V. Gelboin, B.J. Song, S.S. Park and F.K. Friedman, Detection and purification of cytochromes P-450 in animal tissues with monoclonal antibodies, J. Biol. Chem., 259 (1984) 12279-12284.

30 N. Fernandez, M. Roy and P. Lesca, Binding characteristics of Ah receptors from rats and mice before and after separation from hepatic cytosols. 7-Hydroxyellipticine as a competitive an- tagonist of cytochrome P-450 induction, Eur. J Biochem., 172 (1988) 585-592.

31 J.C. Cook and E. Hodgson, Induction of cytochrome P-450 in congenic C57BL/6J mice by isosafrole: lack of correlation with the Ah locus, Chem.-Biol. Interact., 58 (1986) 233-240.

32 A.B. Okey, A.W. Dube and L.M. Vella, Binding of benzoIa]pyrene and dibenz[a,h]anthracene to the Ah receptor in mouse and rat hepatic cytosols, Cancer Res., 44 (1984) 1426-1432.

33 T.H. Zytkovicz, Species and tissue distribution of the 4S carcinogen binding protein and its possible role in cytochrome P-450 induction, Chem.-Biol. Interact., 62 (1987) 15-24.

34 M. Harris, C. Kamps and S. Safe, Role of the 4-5S binding protein in the induction of aryl hydro- carbon hydroxylase in the rat, Carcinogenesis, 9 (1988) 1475 - 1479.

35 W.H. Houser, C.K. Cunningham, R.N. Hines, W.I. Schaeffer and E. Bresnick, Interaction of the 4S polycyclic aromatic hydrocarbon-binding protein with the cytochrome P-450c gene, Arch. Biochem. Biophys., 259 (1987) 215-223.

36 W.H. Houser, R.N. Hines and E. Bresnick, Implication of the '4S' polycyclic aromatic hydro- carbon binding protein in the transregulation of rat cytochrome P-450c expression, Biochemistry, 24 (1985) 7839- 7845.

37 A.B. Okey, L.M. VeUa and P.A. Harper, Detection and characterization of a low affinity form

Page 17

219

of cytosolic Ah receptor in livers of mice nonresponsive to induction of cytochrome P1-450 by 3-methylcholanthrene, Mol. Pharmacol., 35 (1989) 823-830.

38 A. Poland and E. Glover, 2,3,7,8-tetrachlorodibenzo-p-dioxin segragation of toxicity with the Ah locus, Mol. Pharmacol., 17 (1980) 86-92.

39 J.K. Selkirk and M.C. MacLeod, Metabolism and macromolecular binding of benzo[a]pyrene and its noncarcinogenic isomer benzo[e]pyrene in cell culture, in: P.W. Jones and P. Leber (Ed.), Polynuclear Aromatic Hydrocarbons, Ann Arbor Science Publ., Inc., Ann Arbor, 1979, pp. 21-35.

40 M.C. MacLeod, W. Levin, A.H. Conney, R.E. Lehr, B.K. Mansfield, D.M. Jerina and J.K. Selkirk, Metabolism of benzo[e]pyrene by rat liver microsomal enzymes, Carcinogenesis, 1 (1980) 165-173.

41 R.E. Lehr, C.W. Taylor, S. Kumar, W. Levin, R. Chang, A.W. Wood, A.H. Conney, D.R. Thak- ker, H. Yagi, H.D. Mah and D.M. Herina, Differences in metabolism provide a basis for the low mutagenicity and carcinogenicity of benzo[e]pyrene compared to benzo[a]pyrene, in: P.W. Jones and P. Leber (Ed.), Polynuclear Aromatic Hydrocarbons, Ann Arbor Science Publ., Inc., Ann Arbor, 1979, pp. 37-49.

42 J. Jacob, A. Schmoldt and G. Grimmer, Benzo[e]pyrene metabolism in rat liver microsomes: dependence of the metabolite profile on the pretreatment of rats with various monooxygenase inducers, Carcinogenesis, 4 (1983) 905-910.

43 P.M. Tang and J.Y. Chiang, Modulation of reconstituted cholesterol 7 alpha-hydroxylase by phosphatase and protein kinase, Biochem. Biophys. Res. Commun., 134 (1986) 797-802.

44 W. Pyerin, F. Horn and H. Taniguchi, Posttranslational modifications of the cytochrome P-450 monooxygenase system, J. Cancer Res. Clin. Oncol., 113 (1987) 155-159.

45 F.L. Bellino and L. Holben, Placental estrogen synthetase (aromatase): evidence for phosphatase-dependent inactivation, Biochem. Biophys. Res. Commun., 162 (1989) 498-504.

46 I. Jansson, P.M. Epstein, S. Balns and J.B. Schenkman, Inverse relationship between cytochrome P-450 phosphorylation and complexation with cytochrome b5, Arch. Biochem. Biophys., 259 (1987) 441-448.

47 W. Pyerin, H. Tauiguchi, F. Horn, F. Oesch, Z. Amelizad and T. Friedberg, Isoenzyme-specific phosphorylation of cytochromes P-450 and other drug metabolizing enzymes, Biochem. Biophys. Res. Commun., 142 (1987) 885-892.

48 B. Bartlomowicz, T. Friedberg, D. Utesch, E. Molitor, D. Platt and F. Oesch, Regio- and stereoselective regulation of monooxygenase activities by isoenzyme-selective phosphorylation of cytochrome P450, Biochem. Biophys. Res. Commun., 160 (1989) 46-52.

49 W. Pyerin and H. Taniguchi, Phosphorylation of hepatic phenobarbital-inducible cytochromes P-450, EMBO J., 8 (1989) 3003-3010.

50 M.G. Caron, K. Goldstein, K. Savard and J.M. Marsh, Protein kinase stimulation of a reconstituted cholesterol side chain cleavage enzyme system in bovine corpus luteum, J. Biol. Chem., 250 (1975) 5137-5143.

51 G.S. Boyd, A.M. Gorban and M.E. Lawson, Dietary effects on certain adrenal cortical functions in the rat, Nutr. Metab., 24 (1980) 74-89.

52 I. Vilgraln, G. Defaye and E.M. Chambaz, Adrenocortical cytochrome P-450 responsible for cholesterol side chain cleavage (P-450scc) is phosphorylated by the calcium-activated, phospholipid-sensitive protein kinase (protein kinase C), Biochem. Biophys. Res. Commun., 125 (1984) 554- 561.

53 W. Pyerin, C.R. Wolf, V. Kinzel, D. Kubler and F. Oesch, Phosphorylation of cytochrome-P- 450-dependent monooxygenase components, Carcinogenesis, 4 (1983) 573-576.

54 W. Pyerin, H. Taniguchi, A. Stier, F. Oesch and C.R. Wolf, Phosphorylation of rabbit liver cytochrome P-450 LM2 and its effects on monooxygenase activity, Biochem. Biophys. Res. Commun., 122 (1984) 620-624.

55 J.G. Ghazarian and D.M. Yanda, Inhibition of 25-hydroxyvitamin D1 alpha-hydroxylase by renal mitechondrial protein kinaseocatalyzed phosphorylation, Biochem. Biophys. Res. Commun., 132 (1985) 1095-1102.

56 P.L. Iversen and M.R. Franklin, Microsomal cytochrome P-450 'handprints': five fractions from anion-exchange high-pressure liquid chromatography provide a rapid preliminary screen for se-

Page 18

220

57

58

lectivity in the induction and destruction of rat hepatic cytochrome P-450 subpopulations, Tox- icol. Appl. Pharmacol., 78 (1985) 1-9. Y. Funae, R. Seo and S. Imaoka, Multiple forms of cytochrome P-450 from microsomes of rat liver, kidney and lung resolved by high-performance liquid chromatography, Biochem. Int., 11 (1985) 523- 531. X. Ma, J.A. Gibbons and J.G. Babish, Benzo[e]pyrene pretreatment of immature, female C57BL/6J mice results in increased bioactivation of aflatoxin B1 in vitro, Toxicol. Lett., 59 (1991) 51 - 58.