Introduction

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The AHR belongs to the basic helix-loop-helix family of DNA-binding proteins distinct from the zinc finger type steroid receptor proteins (Swanson and Bradfield, 1993; Okey et al., 1994). It regulates transcriptionally the expression of several different drug-metabolizing enzymes including CYP1A1, CYP1A2, glutathione S-transferase (GSTYa), UDP-glucuronosyltransferase (UGT1*06), NAD(P)H:quinone oxidoreductase (NQO₁) and the tumor-associated aldehyde dehydrogenase (ALDH3c) (Landers and Bunce, 1991). It appears also to have an important physiological role during development, as indicated from recent experiments carried out with transgenic Ah receptor-deficient mice, which exhibited immunodeficiency and hepatic fibrosis (Fernandez-Salguero et al., 1995). Upon ligand activation the receptor dissociates from the 90-kdalton heat shock protein and dimerizes with the nuclear basic helix-loop-helix transcription factor ARNT, which shares the PAS region with AHR, also common to the Drosophila regulatory proteins Sim and Per (Takahashi, 1992; Hankinson, 1994). ARNT contains, however, an amino acid sequence recognizing the E box motif CACGTG, which suggests a separate, AHR-independent role for ARNT (Antonsson et al., 1995). The AHR-ARNT heterodimer complex recognizes and binds to specific xenobiotic (or dioxin) responsive elements, initiating transcriptional activation (Whitlock, 1994).

The substituted benzimidazole omeprazole has been shown to increase the expression of CYP1A1 and CYP1A2 in human hepatocytes (Diaz et al., 1990) and in the human alimentary tract (McDonnell et al., 1992). Other benzimidazole derivatives, like the anthelmintic drugs albendazole (Souhaili-el Amri et al., 1988) and oxfendazole (Gleizes et al., 1991), as well as thiabendazole (Aix et al., 1994) also induce the CYP1A subfamily in rodents. The induction mechanism of hepatic CYP1A by the benzimidazoles is not clear. Omeprazole was first found to cause an increased amount of CYP1A mRNA in cells, which suggested transcriptional activation. However, based on competition experiments with [³H]TCDD as an AHR ligand, Daujat et al. (1992) were unable to find any specific binding of omeprazole to the hepatic cytosolic AHR. Similar results were obtained by Aix et al. (1994) with thiabendazole as competitor and by Curi-Pedrosa et al. (1994) with lanzoprazole. By contrast, Quattrochi and Tukey (1993) found that omeprazole triggered the translocation of AHR to the nuclei and its binding to the XRE of the regulatory region of the human CYP1A1 gene.

The group of the AHR-associated drug-metabolizing genes are typically expressed and induced by TCDD or 3 MC predominantly in the centrilobular region of the liver (Baron et al., 1981; Gebhardt, 1992; Oinonen et al., 1994). Understanding how this position-dependent expression and induction is regulated may help to explain why hepatotoxins, that are activated and detoxicated by these gene products, commonly cause zonal damage in the liver. Because both AHR and the nuclear translocator protein ARNT seem to be necessary components in the expression and induction of these genes, elucidation of their acinar distribution was considered important. In previous studies administration of submaximal doses of TCDD, 3MC and NF, all considered as AHR ligands, was shown to result in distinctly different zonal CYP1A1 induction patterns (Bars and Elcombe, 1991; Oinonen et al., 1994). To elucidate this phenomenon further, we investigated how induction by omeprazole, an atypical, probably ligand-independent but AHR-associated compound, was distributed. Zonation was studied by analysis of cell lysates extracted from either the periportal or the perivenous (centrilobular) liver region after site-specific digitonin infusion. We observed a distinct perivenous zonation of AHR mRNA and protein and of omeprazole-dependent induction of CYP1A1, as compared with absent or moderate zonation of ARNT mRNA.

	Methods

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Animals. Male Wistar rats weighing 190 to 250 g and fed rat and mouse No. 1 maintenance diet (Special Diets Service Ltd., Witham, Essex, England) or R3 diet (Astra Ewos, Sweden) were used. For induction experiments, animals were injected daily intraperitoneally with NF (Aldrich Chemical Co. Inc., Milwaukee, WI; 15 or 100 mg/kg), with 3MC (Sigma Chemical Co., St Louis, MO; 5 or 25 mg/kg) or with vehicle (corn oil, 5 ml/kg) for 3 or 4 days. Protein data were analyzed after low-dose induction and mRNA after high-dose induction. A separate set of animals were treated for 4 or 7 days with daily intragastric doses (140 mg/kg) of omeprazole (Astra Hässle AB, Sweden) in Methocel (Sigma, Munich, Germany) solution. The last dose of the chemicals was given 14 to 18 h before liver sampling. The experiments were approved by the local committee for animal experiments.

Antisera. Antiserum against the rat AHR was prepared in rabbits by immunization of conjugates between ovalbumin and a peptide corresponding to amino acids 12 to 31 of the rat AHR. The coupling was carried out via tyrosine linked to the C-terminus of the peptide. This conjugate was mixed with Freund's adjuvant and used for the immunization of rabbits. Immunization and two booster injections were performed using 1 mg of peptide per rabbit. A brief characterization of the sera, which was obtained after the second boost and used as a source of polyclonal antibodies, has appeared (Sadar et al., 1995).

Collection of periportal and perivenous cell lysates. Periportal and perivenous cell lysates were obtained by digitonin infusion (Quistorff and Grunnet, 1987) during in situ perfusion of anesthetized (phenobarbital, 60 mg/kg i.p.) rats as described before (Saarinen et al., 1993). Periportal cells were lysed by a brief (8-12 s) infusion of digitonin (3.5 mM; ICN Chemicals, Cleveland, OH) via the portal vein, and the lysate was collected by immediate retrograde flushing. Subsequently, perivenous cell lysates were obtained by infusing digitonin via the upper vena cava followed by antegrade flushing. The order of digitonin infusions was regularly reversed. The length (penetration depth) of the digitonin pulse was determined empirically to lyse approximately one fourth to one third of the cells along the plate in either the proximal or distal part of the sinusoid. Alanine aminotransferase (EC2.6.1.2.), a periportal marker, was assayed from the lysates to verify the zonal origin.

Preparation of rat liver cytosol. Livers were perfused via the portal vein in PBS, cut into small pieces and carefully homogenized with a Dounce homogenizer in 3 volumes of 10 mM potassium phosphate buffer, pH 7.2, containing 1 mM ethylenediaminetetraacetic acid, 10% glycerol and 2 mM -mercaptoethanol (EPGM-buffer). The homogenate was centrifuged for 60 min at 105,000 × g. Excessive lipids were removed from the ensuing supernatant and the cytosol frozen in small aliquots at 70°C.

Cloning of the rat AH receptor, cell transfection and immunodetection. Total RNA from a control male rat was isolated according to Okayama et al. (1988). RT-PCR cloning of the receptor cDNA was carried out with the forward primer (5 GAC AAG CTT ATG AGC AGC GGC GCC AAC AT 3) and reverse primer (5 CAG TCT AGA CTA CAG GAA TCC GCT GGG TG 3) (first strand synthesis kit from Clontech, Palo Alto, CA). The resulting cDNA was inserted into pCMV4 as described by Thomson et al. (1984), with HindIII and XbaI. COS-1 cells were transfected by a DEAE dextran method as described elsewhere (Johansson et al., 1994). Cells were harvested after 60 h and lysed by sonication. The product was centrifuged at 10,000 × g for 10 min and the supernatant frozen in aliquots.

RT-PCR-based quantification. Relative amounts of AHR and ARNT mRNA in periportal and perivenous cell lysates were compared by a PCR-based semiquantitative method as described previously (Saarinen et al., 1993; Oinonen et al., 1993). Total RNA was isolated from digitonin cell lysates essentially as in Chomzynski and Sacchi (1987) or with Qiagen's RNeasy^TM kit (Qiagen GmbH, Hilden, Germany). First-strand cDNA was produced from 4 µg of total RNA in a 80-µl reaction volume with random hexanucleotide primers and the Promega's Reverse Transcription System (Promega, Madison, WI). For AHR 5 TCC ATG TAC CAG TGC CAG G 3 (forward) and 5 ATA TCA GGA AGA GGC TGG GC 3 (reverse) primers, for ARNT 5 GTC TCC CTC CCA GAT GAT GA 3 (forward) and 5 AAG AGC TCC TGT GGC TGG TA 3 (reverse), and for -actin 5 TGC AGA AGG AGA TTA CTG CC 3 (forward) and 5 GCA GCT CAG TAA CAG TCC G 3 (reverse) primers were used to amplify 212-, 218- and 211-bp fragments, respectively. cDNA (2-5 µl) was amplified in a 100-µl reaction volume containing 2 U Taq DNA polymerase, 1× PCR buffer (both from Boehringer Mannheim, Mannheim, Germany), 25 (-actin), 50 (ARNT) or 100 pmol (ARNT) of both primers, 0.2 mM each dNTP (Promega) and 2.0 (-actin) or 2.5 mM MgCl₂. The hot start procedure of Molecular Bio-Products (San Diego, CA) was used or mineral oil (100 µl; Sigma) was added on top and 20 (-actin) or 26 to 28 cycles, 30 s/94°C, 60 s/55°C (-actin) or 57°C, and 60 s/72°C were run. The last elongation step was extended to 5 min. All PCR reactions were linear up to 28 to 29 cycles. Within each run, linearity was ensured by varying the amount of cDNA. For initial inspection, PCR products were electrophoresed in 4% NuSieve GTG agarose gels (FMC BioProducts, Rockland, ME), the gels were stained with ethidium bromide and were video photographed on an ultraviolet transilluminator. For quantitation, the amplification products were run on anion-exhange high-performance liquid chromatography (Katz and Dong, 1990), as modified by Oinonen and Lindros (1995), with a Perkin Elmer TSK DEAE-NPR column with detection at 260 nm.

Immunoquantification. Samples corresponding to 10 to 40 µg of protein were loaded onto 8.7% sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels. After electroblotting to nitrocellulose filters, incubation was carried out with AHR or CYP1A1 antiserum, the filters were developed with the Amersham ECL Western blotting detection kit and the filters were exposed to Kodak Scientific Imaging film X-OMAT AR (XAR5) film. Each filter contained standard samples. Several different exposures of the films were carried out, and the films were scanned densitometrically by a Molecular Dynamics Personal Densitometer. Linearity with respect to the amount of loaded protein was always assured.

Data analysis. To reduce interseries variation in quantification of PCR products, normalization to -actin was performed. Alternatively, data from high-performance liquid chromatography quantitations of RT-PCR products from four interdependent parameters, AHR, ARNT, GST Ya1 and ALDH 3, were treated as follows. Within each treatment and sample type (periportal or perivenous) group and for each PCR run, the relative deviation of a sample from the mean was calculated. Then for each sample the mean deviation, as observed in the four PCR runs, was calculated and normalization was achieved by dividing with the mean deviation coefficient.

	Results

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Origin of periportal and perivenous cell lysates. To verify the zone selectivity of the cell lysates obtained from either the periportal or perivenous region we used the activity of alanine aminotransferase, which is expressed in a descending portacaval gradient. The activity of alanine aminotransferase in periportal samples was 12.2 ± 5.4 (mean ± S.D., n = 15, series 1) or 16.7 ± 3.4 (n = 7, series 2) higher than perivenous eluates. These results are in full agreement with our previous findings (Oinonen et al., 1993, 1994), indicating complete zone selectivity of the cell lysates.

Characterization of AHR antisera. The specificity of the antibody raised against the synthetic 20-mer peptide was tested in two ways. First, COS-1 cells were transfected with the AHR insert. Supernatants from COS-1 cells transfected with vector alone (pCMV) or with the insert were run together with a sample from rat liver cytosol. Immunostaining of a protein band of the expected size (about 100 kdaltons) was only observed in samples from AHR-transfected COS-1 cells (fig. 1). The mobility of this protein was the same as that of the immunopositive band observed in samples from rat liver cytosol. Second, blocking experiments with the AHR peptide synthesized for immunization (amino acids 12-31) were performed. Samples from the cytosol and from periportal and perivenous cell eluates were run (fig. 2). These experiments also demonstrated that after preincubation with the peptide the 100-kdalton protein band corresponding to the AHR protein disappeared.

View larger version (58K): [in this window] [in a new window]   Fig. 1.   Immunodetection of AHR protein in COS cells transfected with AHR cDNA and in rat liver cytosol. Western blot analysis of protein isolated from COS-1 cells transfected with AHR cDNA inserted into pCMV4 are shown on the left-hand side of the picture. Supernatants (10,000 × g) isolated from cells transfected with vector alone (pCMV) or vector with insert (AHR) corresponding to 20 µg of protein or rat liver cytosol (1 µg of protein) were treated with anti AHR-peptide antiserum. The arrow indicates the position of the receptor protein.

View larger version (50K): [in this window] [in a new window]   Fig. 2.   Antigen-blocked immunodetection of AHR protein. Two identical gels were run with 20 µg of rat liver cytosol and 40 µg of perivenous (PV) or periportal (PP) protein eluates in the respective lanes with 8.7% polyacrylamide gels. After transfer and blocking, the filters were incubated with either AHR-peptide antiserum or with the AHR antiserum that had been incubated for 1 h at room temperature with the peptide (0.3 mg/ml antiserum; 100 µM) used for immunization. The antisera were diluted 1:500 in both cases. Immunostaining was almost undetectable in periportal samples. The filters were developed with Amersham ECL Western blotting detection kit and exposed to Kodak Scientific Imaging film X-OMAT AR (XAR5) film. The arrow indicates the position of AHR.

Acinar distribution of AHR. Zonation of AHR was studied both at the mRNA and the protein level. The most surprising finding was that immunostaining of AHR protein in periportal samples was almost undetectable, in contrast to the strong staining seen in cell lysates from the perivenous region (fig. 3). Quantification of immunodetectable AHR by densitometry revealed an at least 40-fold higher concentration in perivenous as compared with periportal cell lysates (fig. 4). Animals were pretreated with 3MC or NF to investigate whether these AHR ligands would affect the amount and the zonal distribution of AHR. Analysis of samples obtained from these animals showed that neither inducer significantly affected the amount of cytosolic AHR protein and that the perivenous dominance of AHR protein persisted after induction.

View larger version (69K): [in this window] [in a new window]   Fig. 3.   Immunodetection of AHR protein in cell lysates from the periportal and the perivenous region. Cell lysates from the periportal (p) or perivenous (v) liver region obtained from controls (C) or from rats treated with NF or 3MC, corresponding to 40 µg of protein, were loaded in each well. A broad range molecular weight ladder was run on the lane to the right. Periportal samples showed almost undetectable immunostaining of the 100-kdalton size protein (arrow). Detection was performed as described for figure 2.

View larger version (13K): [in this window] [in a new window]   Fig. 4.   Zonal distribution of AHR protein. Effect of pretreatment with 3MC or NF. The figure shows mean values ± S.D. (n = 6) of densitometric scannings of Western blot analyses of cell lysates from the periportal or perivenous region.

View larger version (83K): [in this window] [in a new window]   Fig. 5.   RT-PCR analysis of AHR and ARNT mRNA in periportal and perivenous cell lysates. Ethidium bromide stained RT-PCR products from two pairs of periportal (p) and perivenous (v) samples run on agarose gel electrophoresis (4% NuSieve GTG from FMC Bioproducts) is shown. cDNA was amplified as described under "Materials and Methods," except that 30 cycles were performed. A DNA molecular weight marker (Boehringer VI) was run on the right and contains fragments of 154, 220, 234, 298, 394, 453, 517, 653, 1033, 1230, 1766 and 2176 bp.

View this table: [in this window] [in a new window]   TABLE 1 Zonal distribution of AHR mRNA and ARNT mRNA and the effect of treatment with 3MC, NF or omeprazole RT-PCR products of AHR mRNA or ARNT mRNA from periportal (pp) and perivenous (pv) liver eluates obtained from rats pretreated with 3MC, NF, omeprazole (OME) or vehicle only (control) were quantified by HPLC as described under "Methods." Mean values of normalized peak areas ± S.D. with the number of experiments in parentheses are shown. The mean of the ratio between the amplified product of the corresponding perivenous and periportal sample is given in the last column.

Distribution of ARNT mRNA. In contrast to AHR mRNA, no zonation of ARNT mRNA was observed in the first series (pv/pp = 0.8) and only moderate perivenous zonation (pv/pp = 1.9) in the second series (table 1). None of the ligands affected the zonation, but after 3MC treatment an increased amount of ARNT mRNA was observed.

Distribution of CYP1A1 after induction by omeprazole. To test the functional significance of the perivenous expression of the AHR, in vivo CYP1A1 induction experiments were performed. For this purpose we used omeprazole, a widely used antiulcer drug, that has been shown to cause induction of human CYP1A1 and CYP1A2, evidently via a ligand-independent but AHR-associated mechanism. Administration of omeprazole (140 mg/kg) for 7 days caused significant induction of CYP1A1 protein (fig. 6). Although the degree of induction was modest compared with 3MC, comparison of periportal and perivenous samples demonstrated that most of the induction took place in the perivenous region (fig. 7), which suggested the involvement of AHR in this process.

View larger version (96K): [in this window] [in a new window]   Fig. 6.   Cell lysate CYP1A1 protein after omeprazole treatment. Western blot filters of cell lysates obtained from the perivenous (lanes 1 and 3 from the left) or the periportal (lanes 2 and 4) region and homogenates from livers of omeprazole-treated (lanes 5 and 6) and control (lane 7) rats.

View larger version (10K): [in this window] [in a new window]   Fig. 7.   Zonation of omeprazole-induced CYP1A1 protein. The figure shows mean values ± S.D. (n = 5) of densitometric scannings of Western blot analyses of cell lysates from the periportal (PP) or perivenous (PV) region from omeprazole-treated and control animals.

	Discussion

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In this study the acinar zonation of expression was studied by comparing cell lysates obtained from either the periportal or the perivenous (centrilobular) acinar liver region by region-selective infusion of digitonin solution to the in situ perfused rat liver (Saarinen et al., 1993). Although the digitonin solution efficiently destroys the plasma membrane and also releases proteins from the endoplasmic reticulum, the mitochondrial and nuclear membranes are relatively resistant, because of their lower cholesterol content. Virtually no DNA is detected in these lysates. Thus nuclear proteins most probably are not released by this procedure. As shown previously by us (Bühler et al., 1992; Oinonen et al., 1994), comparison of periportal and perivenous cell lysates gives essentially the same information regarding zonation of either protein or mRNA as that obtained by immunohistochemistry and in situ hybridization experiments. The present technique has the advantage that from one sample several gene products can be analyzed, both at the protein and mRNA level.

We here demonstrate that there is a dramatic regional heterogeneity in the expression of AHR protein in rat liver. The amount of AHR protein in cell lysates from the perivenous region was found to be at least 40-fold higher than in samples from the periportal region. The results based on RT-PCR analysis of the AHR mRNA indicated a similar, but clearly less dramatic perivenous zonation. The apparent difference in the degree of protein and mRNA zonation may reflect a zone-specific posttranslational regulation of AHR protein, but may also be caused at least partly by methodological constrains. Although for the RT-PCR-based mRNA analysis conditions were established to give a linear response, the technique must nevertheless be regarded as semiquantitative. Thus the 2-fold difference in the pv/pp ratio of AHR mRNA in the two separate series (3.5 and 6.3, respectively) is not necessarily be caused by biological variation. However, this difference was not caused by the fact that in series 2 the mRNA values were expressed relative to -actin mRNA, because this procedure did not affect the pv/pp ratio. Considering these limitations the present data nevertheless indicate that a substantial part of the regulation of this remarkable zonation is pretranslational. At this time we have little knowledge of how this zonation is regulated. For instance, is there a sharp or diffuse boundary of AHR expression along the cells within the acinus? Unfortunately, our attempts to answer this question by immunohistochemistry were unsuccessful, possibly because the epitope recognized by the peptide antigen is not exposed in tissue sections. Zonation could be caused by transcriptional activators acting perivenously, but it is also conceivable that in the periportal region proposed Negative Response Elements retard the translational rate or that the degradation of AHR protein is faster.

In functional terms the marked zonation of AHR protein is the most relevant observation. It was recently demonstrated that the expression of two of the XRE-possessing genes, CYP1A1 and UDPGT, was absent or extremely low in transgenic AHR-deficient mice (Fernandez-Salguero et al., 1995). This strongly suggests that a functional AHR is necessary for the constitutive expression of XRE-possessing genes. Consequently, the very low level of AHR expression in the periportal region may explain the limited expression of the so-called AH battery genes in this region. Furthermore, the fact that enzyme induction caused by exogeneous AHR ligands, including dioxin and 3MC, is seen preferentially in the perivenous region, suggests that in the periportal region the low expression of AHR prevents or limits enzyme induction.

Our previous observation, that induction of CYP1A1, 1A2 mRNA and protein by NF exhibits a deviant periportal expression (Oinonen et al., 1994) is not readily explained by the present data. Both 3MC and NF treatment resulted in a significant 2- to 2.5-fold induction of AHR mRNA, but no significant induction of AHR protein was observed. Furthermore, the marked perivenous expression pattern remained regardless of the inducer. Although in the normal liver AHR protein is located mainly in the cytosol, it is conceivable that upon ligand stimulation by 3MC or NF some translocation into the nucleus could occur. This fraction of the AHR protein would go undetected with the present sampling technique and could therefore explain the apparent absence of increase in AHR protein upon ligand stimulation.

AHR also seems to be involved in the regulation of several important growth-regulatory proteins, including the epidermal growth factor receptor, interleukin 1 and transforming growth factors  and  (see Okey et al., 1994). Interestingly, a marked zonation of the epidermal growth factor receptor has been described (Marti and Gebhardt, 1991), which suggests a functional association with the zonal distribution of AHR. This suggestion needs to be verified by studying the zonation of other growth factors as well.

Compared with AHR, ARNT mRNA is much less zonated. Provided that this holds for the distribution of the corresponding protein, this suggests that ARNT does not determine the zonated expression of XRE-possessing genes. The different zonation of the structurally related AHR and ARNT is compatible with their different regulation, as also illustrated by their different subcellular localization (Pollenz et al., 1994). ARNT contains an amino acid sequence recognizing the E box motif CACGTG, which suggests a separate, AHR-independent role for ARNT (Antonsson et al., 1995). ARNT probably dimerizes with other transcriptional factors. Such factors might have a periportal hepatic distribution.

The involvement of AHR in the omeprazole-dependent induction of CYP1A1 is still under debate. Although omeprazole has been found not to possess normal AHR ligand-binding properties, stimulation of hepatoma cells with the drug has been shown to cause translocation of the receptor to the nucleus and subsequent binding to XRE (Quattrochi and Tukey, 1993). In the present investigation we demonstrate that treating animals with omeprazole results in CYP1A1 induction mainly in the same hepatic region where AHR is constitutively located. This provides further evidence for a role of AHR also in the omeprazole-dependent activation, although this process occurs via a mechanism that does not involve specific binding to the receptor. Evidence for a ligand-independent action was recently obtained from experiments describing that signals generated through release of human keratinocyte cells from cell substratum caused activation of CYP1A1 expression in the absence of AHR ligands (Sadek and Allen-Hoffman, 1994a) and that suspension of mouse hepa 1c1c7 cells for 4 h caused induction of CYP1A1 mRNA and activation of the murine AHR to an XRE-binding form in the absence of any AHR ligands added (Sadek and Allen-Hoffman, 1994b). The involvement of a receptor-coupled intracellular signal transduction system has been proposed (Lesca et al., 1995; Johansson et al., 1995), but the detailed mechanisms behind this receptor activation require further investigations.

In conclusion, our data demonstrate a selective centrilobular hepatic expression of AHR in rat liver. Because a functional AHR seems a prerequisite for normal expression of several AHR-associated genes, our observation may explain why many of these genes are expressed mainly in the centrilobular region, both under constitutive and induced conditions. The molecular factors responsible for the zone-restricted expression of AHR remain unknown, but constitute a challenging target for future research.