−) Transgenic Mice

ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS

Vol. 349, No. 2, January 15, pp. 349–355, 1998 Article No. BB970465

The 4S Benzo(a)pyrene-Binding Protein Is Not a Transcriptional Activator of Cyp1a1 Gene in Ah Receptor-Deficient (AHR 0/0) Transgenic Mice Julie Foussat, Philippe Costet, Pierre Galtier, Thierry Pineau, and Pierre Lesca1 Laboratoire de Pharmacologie et Toxicologie, Institut National de la Recherche Agronomique, BP3, 31931 Toulouse, France

Received August 13, 1997, and in revised form October 8, 1997

In an effort to better understand the role of the 4S benzo(a)pyrene-binding protein in the induction of CYP1A1 by PAHs, we used a genetically engineered mouse line deficient in Ah receptor (AHR 0/0). First, we demonstrated through binding experiments analyzed by sucrose gradient sedimentation and gel permeation chromatography that AHR 0/0 mice have no detectable AHR protein. In contrast, this AHR-deficient line expressed a 4S protein which efficiently binds BP as it does in hepatic cytosol from C57BL/6 mice. In vivo BP exposure in AHR-deficient mice proved the inability to sustain any CYP1A1 mRNA or CYP1A1 protein induction. These findings demonstrate the requirement of an active AHR to sustain the transactivation pathway leading to CYP1A1 induction. Surprisingly, the 4S BP-binding protein, which was previously characterized as the glycine N-methyltransferase, was completely devoid of such an enzymatic activity after purification by Sephacryl gel permeation chromatography. Moreover, sedimentation and chromatographic experiments, under nondenaturing conditions, do not support the assumption of 4S protein as a subunit of a multimeric protein (GNMT) displaying a molecular mass of 150 kDa. q 1998 Academic Press Key Words: Ah receptor; 4S protein; induction; glycine N-methyltransferase; knockout mice.

In nonmammalian and mammalian species, polycyclic aromatic hydrocarbons (PAHs),2 such as 3-methyl1 To whom correspondence should be addressed. Fax: 33(0)5-6128-53-10. 2 Abbreviations used: PAHs, polycyclic aromatic hydrocarbons; 3MC, 3-methylcholanthrene; BP, benzo(a)pyrene; AHR, aryl hydrocarbon receptor; HAH, halogenated aryl hydrocarbon; TCPOBOP, 1,4-bis[2-(3,5-dichloropyridyloxy)]-benzene; Hepes, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid; AHH, aryl hydrocarbon hy-

cholanthrene (3-MC) and benzo(a)pyrene (BP), and halogenated aromatic hydrocarbons (HAHs), such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), transcriptionally induce the genes of the Cyp1 family of cytochrome P450s, leading to an increase of the following proteins: CYP1A1, CYP1A2, CYP1A3, and CYP1B1 (1,2 for reviews). A sophisticated signal transduction pathway between some prototypic inducers (TCDD, 3-MC) and their nuclear targets has been established. It involves a basic helix–loop–helix domain-containing cytosolic receptor named aryl hydrocarbon receptor (AHR). This three-step pathway includes: (i) cytosolic activation of the quiescent form of the receptor by its ligand and subsequent release of two chaperone proteins (heat shock protein 90) (3) and newly identified associated proteins (4, 5), (ii) pairing of the activated receptor with its nuclear heterodimeric partner (Ah receptor nuclear translocator) (6), and (iii) interaction of the resulting complex with core sequences (xenobiotic response elements) located in the 5* regulatory region of target genes (7). Regarding several molecules of the PAH and HAH families of inducers, the involvement of the AHR in the induction of the Cyp1a1 gene has been established (8). Recently, several non-PAH, non-HAH molecules have been proven to be Cyp1a1 gene activators. Even though these chemicals are inefficient in competing with TCDD for binding to the AHR (9–14), it has been proven, using AHR-deficient mice, that the AHR is fully required to mediate Cyp1a1 gene induction (14, 15). In addition, it is known that the PAH-binding proteins constitute a group with several members like the 4S droxylase; GNMT, glycine N-methyltransferase; EROD, ethoxyresorufin O-deethylase; TCDD, 2,3,7,8-tetrachlorodibenzo-p-dioxin. 349

0003-9861/98 $25.00 Copyright q 1998 by Academic Press All rights of reproduction in any form reserved.

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(16–19) and 8S (20) proteins. Based on these observations one can imagine new signal transduction pathways leading to CYP1A1 induction with no involvement of the AHR. This was proposed regarding BP, a PAH molecule, demonstrated to be ligand of the AHR (21). Following the controversial identification of the 4S protein as the monomer of the tetrameric glycine N-methyltransferase (22–24), it was proposed that this protein would sustain the BP induction of CYP1A1 independently from the AHR (25–27). However, several pieces of evidence have been reported which are not in good accordance with this assumption (2). In this paper we demonstrate, in mouse, that the 4S protein is distinct from glycine N-methyltransferase, and we used a genetically engineered mouse line deficient in AHR (28) to establish that expression of the AHR is a requirement for CYP1A1 induction by BP. MATERIALS AND METHODS Chemicals. [G-3H]Benzo(a)pyrene (80 Ci/mmol), radiochemical purity 97.3%, was purchased from Amersham Corp. (Buckinghamshire, England). Unlabeled TCDF and [3H]TCDD (34.7 Ci/mmol), radiochemical purity ú97%, were purchased from Chemsyn Science Laboratories (Lenexa, KS). Radioactive S-adenosyl-L-[methyl-3H]methionine (80 Ci/mmol), radiochemical purity ú99%, was obtained from Isotopchim (Ganagobie-Peyruis, France). Unlabeled BP, unlabeled S-adenosyl-methionine, Hepes, dextran (Mr 150,000), activated charcoal, cytochrome c, bovine serum albumin, dithiothreitol, and dimethyl sulfoxide were from Sigma Chemical Co. (St. Louis, MO). Ethoxyresorufin was from Pierce Europe (Oud-Biejerland, The Netherlands). Sucrose (density gradient grade) was from BDH Ltd. (Poole, England). Sephacryl S-300 HR was from Pharmacia. TRIzol reagent was from Life Technologies (Cergy-Pontoise, France). 1,4-Bis[2-(3,5dichloropyridyloxy)]-benzene, purity 99.9%, was synthesized according to Poland et al. (29). Polyclonal (mouse) anti-Ah (dioxin) receptor antibody was purchased from Affinity Bioreagents, Inc. (Golden,CO). Anti-mouse IgG (whole molecule) peroxidase conjugate was obtained from Sigma Chemical Co. P4501A antibody, which recognized the P4501A1 and the P4501A2 isoforms, was kindly given by Dr. Franck Gonzalez. All other chemicals were purchased from commercial sources. Animals. C57BL/6 mice (7 weeks old) were from Iffa-Credo (Les Oncins, France). Genetically engineered AHR-deficient (AHR 0/0) mice were from our laboratory. The homozygous AHR 0/0 mice used in this study (28) were selected following several rounds of cross breeding with C57BL/6 males. Therefore, they are predominantly of C57BL/6 genetic background. For this reason this line, the closest from our transgenic line, was selected as the line for the control animals in our experiments. Mice pretreated with TCPOBOP received 3 mg/kg (ip) of the compound dissolved in sunflower oil and they were euthanized 72 h following injection. Treatment of animals. Male C57BL/6 and AHR 0/0 mice received, by intraperitoneal route, a single treatment of benzo(a)pyrene (50 or 100 mg/kg body wt) dissolved in sunflower oil. The mice were sacrificed either 20 or 72 h after treatment. Buffers. The standard buffer used for preparation of cytosol and for binding experiments was 25 mM Hepes, 1.5 mM EDTA, 1 mM dithiothreitol, and 10% glycerol (by vol), pH 7.6 (HEDG). Microsomes were prepared (see below) in 100 mM potassium phosphate, 100 mM Tris, 100 mM KCl, 1 mM EDTA buffer, pH 7.4, containing 20 mM hydroxytoluene (buffer A) and kept at 0807C in 100 mM potassium phosphate, 0.1 mM EDTA, and 20% glycerol, pH 7.4 (buffer B). Preparation of cytosol. Untreated mice were sacrificed by cervical dislocation. Their livers were perfused, in situ, with 0.9% NaCl via

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a needle inserted into the heart and then into the vena cava. After perfusion, the liver was removed, rinsed with 0.9% NaCl, then with HEDG buffer and homogenized in HEDG buffer (3 ml/g liver) with a Teflon/glass homogenizer. All procedures were performed at 47C. The homogenate was centrifuged at 9000g for 20 min and the supernatant was centrifuged at 105000g for 1 h. The cytosolic fraction was collected carefully without disturbing the surface lipid layer or the microsomal pellet. Cytosolic fractions were stored at 0807C for periods of up to 3 months before use in binding experiments or component separation procedure; no loss of activity occured under these conditions. Preparation of microsomes. Benzo(a)pyrene-treated mice were euthanized by cervical dislocation. Livers were perfused with phosphate-buffered saline and then homogenized in buffer A as described above. The homogenate was centrifuged at 9000g for 20 min, and the resulting supernatant was centrifuged at 105,000g for 1 h. The cytosol was removed carefully and the microsomal pellet was washed in buffer A and centrifuged again at 100,000g for 30 min. Microsomes were resuspended in buffer B and stored at 0807C. Separation of binding components by gel permeation chromatography. The separation of binding components was carried out from either the crude cytosol or the 9S-enriched fraction of mouse cytosol. The 9S-enriched fraction was prepared, as described previously (30), in 5–20% sucrose gradients centrifuged for 2 h 40 min in a vertical rotor at 372,000g. The samples of crude cytosol or 9S-enriched fraction, incubated with 20 nM [3H]TCDD and 20 nM [3H]BP for 1 h at 47C and then treated with dextran–charcoal (10 mg charcoal/ml), were loaded onto a column of Sephacryl S-300 HR equilibrated with HEDG buffer. The samples were eluted with the same buffer under gravity flow (5 to 16 ml/h according to the column); 120 fractions (3 ml per fraction) were collected for each sample and were counted for radioactivity. Binding experiments. The standard binding experiments were carried out by incubating 1 ml cytosol or separated cytosolic component with either 20 nM [3H]TCDD or 25 nM [3H]BP, in the absence or presence of competitor, for 1 h at 47C. The radioligands were added to samples in 10 ml dimethyl sulfoxide. When the samples were treated with dextran–charcoal they were added to a dextran– charcoal pellet (10 mg charcoal/ml) pelleted from HEDG–dextran (1 mg/ml) buffer. Dextran–charcoal was resuspended in the sample on a Vortex mixer, incubated for 15 min in the ice, and then removed by centrifugation at 4000g for 15 min. Samples (300 ml) were layered onto either 5–20 or 10–30% linear sucrose density gradients prepared in HEDG buffer. Gradients (4.8 ml) were centrifuged at 47C for 2 h, in a vertical-tube rotor (Beckman VTi 65) at 372,000g. After centrifugation, 22 fractions (232 ml; eight drops per fraction) were collected with a Beckman Recovery System. Radioactivity in each fraction was determined by liquid scintillation counting (Ready Protein from Beckman). Western blot analyses. Microsomal protein concentration was determined by the method of Lowry (31) using bovine serum albumin as the standard. Microsomal samples (5 to 7.5 mg protein), cytosolic fractions, or Ah receptor-enriched samples (15 to 100 mg protein) were resolved on sodium dodecyl sulfate–polyacrylamide (10 or 8.5%) gels and transferred electrophoretically to nitrocellulose filters. Cytochrome P450s 1A1 and 1A2 were detected using the ECL reagent (Amersham, Les Ulis, France).Quantitation was obtained by autoradiograms scanning on a XRS Scanner driven by a Bioimage application analyzer (Roissy, France). Ah receptor was immunologically detected by using polyclonal anti-Ah receptor antibody, raised against a synthetic polypeptide. Northern blot analyses. RNA samples were prepared using the TRIzol reagent according to the manufacturer’s recommandations; 100 mg of fresh liver (large lobe) was dissociated in 1 ml of reagent using a 7-mm-diameter homogenizer (IKKA, Jena, Germany) in 2ml tubes. Twenty micrograms of each sample was resolved on 1.2% agarose-formaldehyde denaturing electrophoresis gels and transferred onto nylon membranes (Gene Screen/, Dupont, Les Ulis,

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France). Random priming radiolabeled probes were hybridized overnight in a dextran–sulfate-containing buffer at 427C (32). Enzyme assays. Ethoxyresorufin O-deethylase (EROD) and benzo(a)pyrene hydroxylase (AHH) activities were determined fluorometrically in microsomes by the methods of Lake (33) and Nebert and Gelboin (34), respectively. GNMT was determined as described by Cook and Wagner (35).

RESULTS

Demonstration of the Absence of the Ah Receptor in AHR 0/0 Transgenic Mice Incubation of hepatic cytosol from C57BL/6 mice with [3H]TCDD leads to the formation of two binding complexes which are found, after centrifugation in 10– 30% sucrose gradient, in the 9S region (near fraction 11 in our assay) and in the upper part of the gradient (Fig. 1). The latter complex had been identified as the result of the [3H]TCDD interaction with lipoproteins

FIG. 2. Gel permeation chromatography of liver cytosols from C57BL/6 and AHR 0/0 mice incubated with [3H]TCDD and [3H]BP. Six milliliters of cytosols (20 mg protein/ml) was incubated with 20 nM [3H]TCDD and 20 nM [3H]BP for 1 h at 47C. Following dextrancoated charcoal adsorption the samples were chromatographed on Sephacryl S-300 HR columns (80 1 2.5 cm) as described under Materials and Methods. Identification of the four peaks has been previously described by Lesca et al. (30, 20).

FIG. 1. Sucrose density gradient analysis of [3H]TCDD binding with liver cytosol from C57BL/6 and AHR 0/0 mice. Liver cytosols (10 mg protein/ml) from C57BL/6 or AHR 0/0 mice were incubated, for 1 h at 47C, with 20 nM [3H]TCDD. Following dextran-coated charcoal treatment as described in the text, 300-ml samples were placed onto a 10–30% sucrose density gradient and the gradients were centrifuged for 2 h at 372,000g. Fractions were collected and radioactivity was counted as described under Materials and Methods.

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(30, 36). Regarding the second peak, many authors have demonstrated that the complex found in the 9S region is the result of the specific binding of the potent inducer [3H]TCDD to the Ah receptor (37–39). Figure 1 shows that after incubation of hepatic cytosol from a male AhR 0/0 mouse with 20 nM [3H]TCDD no radioactive peak is detectable in the 9S region of the sucrose gradient. An identical experiment carried out with female AHR 0/0 mouse displays identical negative responses (data not shown). [3H]TCDD or [3H]BP binding to the various cytosolic components of C57BL/6 mouse can also be examined after gel permeation chromatography of cytosol (Fig. 2). Radioligands bind to four major components: peak I, which is eluted in the void volume, has been identified as the complex of the set of different classes of lipoproteins with both radioligands (30, 36); peak II

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Effects of Benzo(a)pyrene Administration on P4501A Expression

FIG. 3. Western blot illustrating the lack of Ah receptor in the liver cytosol of AHR 0/0 mice. Fractions (70–80) from gel permeation chromatography of liver cytosols from TCPOBOP-treated or untreated C57BL/6 mice and AHR0/0 mice (see Fig. 2) were pooled and concentrated to 1.4, 1.8, and 0.72 mg protein/ml, respectively; 15 mg (TCPOBOP-treated C57 mice), 72 mg (untreated C57 mice), and 40 mg (untreated AHR 0/0 mice) were separated by SDS–polyacrylamide (8.5%) gel electrophoresis and analyzed for Ah receptor expression by immunoblotting as described under Materials and Methods.

The ability of C57BL/6 and AHR 0/0 mice to respond to benzo(a)pyrene was investigated by treatment of animals with two doses (50 or 100 mg/kg body wt) of PAH administered for 20 or 72 h. Northern blot analysis of RNA isolated from livers is presented in Fig. 5A. As expected, a strong induction of CYP1A1 mRNA was observed in the liver of C57BL/6 mice treated with 50 mg/kg for 20 h. In contrast, no induction occured in AHR 0/0 mice under the same conditions, nor after 72 h of treatment with 100 mg/kg BP. We then analyzed the effect of BP on the induction of P4501A1 protein and its enzymatic activities. Western blot analysis demonstrates (Fig. 5B) a large accumulation of P450 1A1 protein in C57BL/6 mouse liver and a complete

(fractions 70–80), which preferentially binds [3H]TCDD corresponds to the Ah receptor (40); peak III (fractions 90–100) and peak IV (fractions 102–116) bind [3H]BP and have been characterized as the 8S protein (20) and the 4S protein (18, 40), respectively. It is clear, from Fig. 2, that the hepatic cytosol from AHR 0/0 mice does not contain the Ah receptor (no peak in fractions 70–80). The pooled and concentrated fractions 70–80 from C57BL/6 mice pretreated with TCPOBOP, or untreated, as well as those from AHR 0/0 mice, were analyzed by Western blotting for expression of the AHR protein (Fig. 3). The expected 97-kDa AHR immunoreactive band was strongly detected in C57BL/6 mice pretreated with TCPOBOP and detected to a lesser extent in untreated mice. In contrast, the AHR 0/0 mice have no AHR protein detectable. The Western blotting of cytosols and AHR-enriched 9 S fractions from the same mice gave exactly the same results (data not shown). Detection and Characterization of the 4S [3H]Benzo(a)pyrene-Binding Protein in AHR 0/0 Mice As shown in Fig. 2, it appears that the expression of the 4S protein (fractions 102–116) is not modified after the disruption of the Ah receptor gene in AHR 0/0 mice, as evidenced by the characterization of the protein incubated with [3H]BP, by sucrose gradient sedimentation assays, either in the crude cytosol (not shown) or after chromatographic separation on Sephacryl gel (Fig. 4). As described previously (20), the BP-binding 8S protein (fractions 90–100) displays a high binding capacity but a low binding affinity for the ligand. Due to the charcoal treatment, which strips the radioligand from its target prior to chromatography on Sephacryl gel, the 8S protein is not easily detectable.

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FIG. 4. Characterization of the liver 4S BP-binding protein in AHR 0/0 mice. Fractions 102–116 from gel permeation chromatography of liver cytosols from C57BL/6 and AHR 0/0 mice (see Fig. 2) were pooled and concentrated, and then were incubated, for 1 h at 47C, with 25 nM [3H]BP. Following dextran-coated charcoal treatment, 300-ml samples were placed onto 5–20% sucrose density gradients and the gradients were centrifuged for 2 h at 372,000g. Fractions were collected and radioactivity was counted as described under Materials and Methods.

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4S PAH-BINDING PROTEIN IS NOT AN ACTIVATOR OF CYP1A1 GENE TABLE II

GNMT Activity of Various Fractions from C57BL/6 and AHR 0/0 Mice

Fraction C57BL/6 mice Cytosol 9S fractionb 4S fractionb 102–116 fractiond (4S protein) 90–100 fractione (8S protein) AHR 0/0 mice 9S fractionb 90–100 fractione (8S protein)

Protein (mg/ml)

GNMTa (nmol/min/mg)

17.0 2.0 4.5 4.0 3.7

0.55 2.92 ndc ndc 1.77

1.1 3.6

3.40 3.07

a Glycine N-methyltransferase. The assays were carried out as indicated under Materials and Methods. The results are representative of three experiments. b Fraction obtained from sucrose gradient sedimentation. c nd, not detectable. d Pool of fractions 102–116 from Sephacryl S-300 chromatography (Fig. 2). e Pool of fractions 90–100 from Sephacryl S-300 chromatography (Fig. 2).

FIG. 5. Effect of benzo(a)pyrene on the expression of cytochrome P4501A1 and 1A2 in C57BL/6 and AHR 0/0 mice. Three male C57BL/6 and AHR 0/0 mice were treated with 50 mg/kg BP during 20 h. The animals were killed and the liver total RNA as well as the liver microsomes were prepared as described under Materials and Methods. (A) Northern blot analysis. RNA (20 mg) was submitted to electrophoresis on a 1.2% agarose gel, transferred to a nitrocellulose membrane (Gene Screen/), and probed with radiolabeled P4501A2 probe (cross hybridization 1A1/1A2). (B) Western blot analysis. Microsomal proteins (4.5 mg) were submitted to a SDS–polyacrylamide (8.5%) gel electrophoresis, transferred to nitrocellulose membrane, and probed with antibodies prepared against mouse liver cytochrome P4501A1.

absence of this P450 in AHR 0/0 mice. Similarly, BP induced EROD and AHH activities in liver microsomes of C57BL/6 mice but not in AHR 0/0 mice (Table I). It can be noted that the P450 1A2 protein as well as the

TABLE I

Monooxygenase Activities of Liver Microsomes from C57BL/6 and AHR 0/0 Mice Treated with Benzo(a)pyrene

Mice

AHRa (pmol/min/mg)

ERODb (pmol/min/mg)

Untreated C57BL/6 Treated C57BL/6 Untreated AHR 0/0 Treated AHR 0/0

299 2097 93 194

392 1020 63 77

Note. The assays were carried out as indicated under Materials and Methods. The results are representative of at least two experiments. a Aryl hydrocarbon hydroxylase. b Ethoxyresorufin O-deethylase.

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EROD and AHH activities are lower in AHR 0/0 mouse liver than in the liver from untreated C57BL/6 animals. Nevertheless, a very slight induction of P4501A2 occurs in AHR 0/0 mice treated with BP, compared to untreated AHR 0/0 mice. This should be further substantiated in specifically designed experiments including a number of animals sufficient to support statistical analysis. 4S and 8S Proteins Lack Any GNMT Activity Glycine N-methyltransferase (GNMT) activity was measured in various fractions obtained from either C57BL/6 or AHR 0/0 mouse cytosols. The results presented in the Table II indicate that GNMT was detected in the 9S fraction prepared by sucrose gradient sedimentation of cytosols, as well as in the 90–100 fractions eluted from Sephacryl S-300 HR chromatography which also contain 8S protein (Fig. 2). Nevertheless, enzymatic assays carried out with purified rabbit liver 8S protein, obtained either by ion-exchange chromatography or by pyrene-Sepharose affinity chromatography (manuscript submitted), indicate that GNMT activity is not associated with 8S protein. Finally, the 4S PAHbinding protein, prepared by sucrose gradient sedimentation or by Sephacryl gel permeation chromatography (fractions 102–116), was completely devoid of GNMT activity (Table II). DISCUSSION

In their original publication (28), Fernandez-Salguero et al. clearly established that following the loss

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of the coding region of the first exon of the mouse AHR gene, the resulting knockout animal did not express any specific mRNA coding for the Ah receptor. Furthermore, using this AHR-deficient mouse line, we performed [3H]TCDD and [3H]BP binding experiments demonstrating that, despite the lack of a functional AHR, several cytosolic PAH-binding proteins remain expressed in these mice. Among them, the 4S protein is expressed in the liver cytosol and can efficiently bind BP as it does in hepatic cytosol from C57BL/6 used as controls. This establishes that the gene encoding the 4S protein is distinct from the AHR gene, which is disrupted in the deficient line. The 8S protein, another PAH-binding protein, is also expressed in AHR-deficient hepatic cytosols. In vivo BP exposure in AHR-deficient mice proved unable to sustain any CYP1A1 induction. This finding demonstrates the requirement of an active AHR to sustain the transactivation pathway leading to CYP1A1 induction. Alone, the active 4S protein, also referred to as GNMT (22), failed to induce CYP1A1 in response to BP in an alternate AHR-independent pathway, as opposed to what was proposed in previous reports (22, 23). In agreement with this observation is the demonstration of the in vivo direct binding of [3H]BP to the AHR under conditions where CYP1A1 is induced (Fig. 6). Four hours after radiolabeled BP injection to wildtype mice, chromatography experiments revealed an hepatic-liganded AHR. Although we proved that BP induction of CYP1A1 requires an active AHR, the need of a functional 4S protein cannot be ruled out. It binds BP with high affinity (26) and it was found to be associated with a regulatory cis element of rat CYP1A1 gene (41–43) and therefore might play a cooperative role in this specific transcriptionnal induction. Considering the severe phenotype resulting from AHR-gene disruption in knockout mice, it could be considered that impairment of physiological conditions in these animals does not allow the CYP1A1-inducing activity of a normal 4S protein. Nevertheless, this hypothesis is unlikely considering the example of the DBA/2 mice, in which no pathological phenotype is observed while a normal 4S protein is unable to induce CYP1A1 due to the expression of an original AHR with severely reduced PAH/HAH binding capabilities (2). Surprisingly, the protein that sediments in the 4S region of sucrose gradients and binds BP efficiently, and that we partially purified by Sephacryl chromatography, is not active in catalyzing GNMT activity, whereas total cytosolic extract and fractions other than 4S, from the same preparation, sustain this activity. Therefore, in accordance with recent reports in rat (22, 44) we questioned the identification of the 4S protein as the glycine N-methyltransferase (22, 23) by Raha et al. From their purification procedure (22) using a Sephacryl S-200 matrix it was concluded that the 4S

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FIG. 6. Elution profile of the various BP-binding components in liver cytosol from [3H]BP-treated C57BL/6 mice, analyzed upon Sephacryl S-300 HR gel permeation chromatography. Nine milliliters (90 mg protein) of liver cytosol from three C57BL/6 mice treated, during 4 h, with 1 mg [3H]BP (143 mCi) was applied to the Sephacryl S-300 column (90 1 1 cm) equilibrated with HEDG buffer, then eluted as described under Materials and Methods. Identification of the four peaks has been previously described by Lesca et al. (30, 20).

protein and GNMT were copurified. Recently, Ogawa et al. demonstrated that (i) GNMT and 4S protein could be separated by Sephacryl S-200 gel permeation chromatography and (ii) 4S protein does not cross-react with the anti-GNMT antibody. In our hands, using a S-300 HR matrix with enhanced resolution, the comparison of chromatographic and GNMT activity data reveals that GNMT does not segregate with 4S protein (Table II). Interestingly this activity is found within fractions containing the 8S protein, a BP-binding protein recently characterized with a large binding capacity but low binding affinity (20). Further purification steps in progress in our laboratory revealed that the purified 8S protein does not exhibit any GNMT activity excluding, and hence the hypothesis of the identity of 8S and GNMT proteins (manuscript submitted). In conclusion, the assumption that the 4S BP-binding protein would be the monomeric subunit of a multimeric protein that displays a molecular weight of 150 kDa under nondenaturing conditions (22, 45) is

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questionable. Chromatographic experiments and sedimentation gradients under nondenaturing conditions reported in this communication do not support this conclusion. As evoked by Raha et al. (22) it might be possible that substantial homology in peptide sequence between monomeric GNMT protein and 4S protein leads to the controversial conclusion of their identity. Regarding the PAH responsive-gene induction, which is of great relevance in toxicology, using genetically engineered animal models, we demonstrate that BPCYP1A1 induction in mice requires the expression of an active AHR. ACKNOWLEDGMENTS This work was supported by grants from the Centre National de la Recherche Scientifique (No. 96/C/04), the Association pour la Recherche sur le Cancer (No. 1386), and the Fondation pour la Recherche Me´dicale. The transgenic mouse line and the antibody raised against P4501A were generous gifts from Dr. F. Gonzalez.

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