Comparative induction of CYP1A1 expression by pyridine and its metabolites.

We compared pyridine and five of its metabolites in terms of (i) in vivo induction of CYP1A1 expression in the lung, kidney, and liver in the rat and (ii) in vitro binding to, and activation of, the aryl hydrocarbon receptor (AhR) in cytosol from rat liver or Hepa1c1c7 cells. Following a single 2.5 mmol/kg ip dose of either pyridine, 2-hydroxpyridine, 3-hydroxypyridine, 4-hydroxypyridine, N-methylpyridinium, or pyridine N-oxide, CYP1A1 activity (ethoxyresorufin O-deethylase), protein level (as determined by Western blotting), and mRNA level (as determined by Northern blotting) were induced by pyridine, N-methylpyridinium, and pyridine N-oxide in the lung, kidney, and liver. The induction by N-methylpyridinium or pyridine N-oxide was comparable to or greater than that by pyridine in some tissues. 2-Hydroxypyridine and 3-hydroxypyridine caused tissue-specific induction or repression of CYP1A1, whereas 4-hydroxypyridine had no effect on the expression of the enzyme. Pyridine and its metabolites elicited weak activation of the aryl hydrocarbon receptor in a gel retardation assay in cytosol from rat liver but not Hepa 1c1c7 cells. However, the receptor activation did not parallel the in vivo CYP1A1 induction by the pyridine compounds, none of which inhibited binding of ¿(3)H2,3,7, 8-tetrachlorodibenzo-p-dioxin to AhR in a competitive assay in rat liver cytosol. The findings are consistent with a role of pyridine metabolites in CYP1A1 induction by pyridine but do not clearly identify the role of aryl hydrocarbon receptor in the induction mechanism.

Physicochemical differences in the AH receptors of the most TCDD-susceptible and the most TCDD-resistant rat strains.

Long-Evans rats (strain Turku AB; L-E) are at least 1000-fold more sensitive (LD50 about 10 microg/kg) to the acute lethal effects of 2, 3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) than are Han/Wistar (Kuopio; H/W) rats (LD50 > 9600 microg/kg). The AH receptor (AHR) is believed to mediate the toxic effects of TCDD and related halogenated aromatic hydrocarbons. We compared the AHRs of L-E and H/W rats to determine if there were any structural or functional receptor differences that might be related to the dramatic difference in the sensitivity of these two strains to the lethal effects of TCDD. Cytosols from liver and lung of the sensitive L-E rats contained about twofold higher levels of specific binding sites for [3H]TCDD than occurred in H/W rats; the Kd for binding of [3H]TCDD to AHR in hepatic cytosols was similar between the two strains. Addition of the oxyanions, molybdate or tungstate (20 mM), had little effect upon ligand binding to AHR in hepatic cytosols from L-E rats whereas in cytosols from H/W rats these agents substantially diminished or totally abolished TCDD binding. The AHR in H/W cytosols also lost ligand-binding function when NaCl (20 to 400 mM) was added to the buffer whereas, in cytosols from L-E rats, the addition of 400 mM NaCl caused the receptor complex to shift from 9S to 6S during velocity sedimentation but did not destroy ligand binding function. AHR from hepatic cytosol of both the L-E and H/W rats could be transformed to the DNA-binding state in the presence of TCDD or other dioxin congeners as assessed by gel mobility shift assays. The most dramatic difference in AHR properties between L-E and H/W rats is molecular mass. Immunoblotting of cytosolic proteins revealed that the AHR in L-E rats has an apparent mass of approximately 106 kDa, similar to the mass of the receptor previously reported in several other common laboratory rat strains. In contrast, the mass of the AHR in H/W rats is approximately 98 kDa, significantly smaller than the mass of receptor reported in any other rat strains. F1 offspring of a cross between L-E and H/W rats expressed both the 106- and the 98-kDa protein. There was no apparent difference in the mass of the AHR nuclear translocator protein (ARNT) between the two strains, but the hepatic concentration of ARNT was about three times as high in L-E as in H/W rats. It will be interesting to find out how the altered structure of the AHR in H/W rats is related to their remarkable resistance to the lethal effects of TCDD.

Prolonged depletion of AH receptor without alteration of receptor mRNA levels after treatment of cells in culture with 2,3,7,8-tetrachlorodibenzo-p-dioxin.

Previous experiments have shown that the total cellular content of the AH receptor (AHR) drops rapidly after exposure of mouse hepatoma cells (Hepa-1) to the potent AHR ligand 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD); within 6 hr after treatment, less than 20% of the original cell content of AHR can be detected by radioligand binding or by immunoblotting. The goals of our current study were to determine the duration of receptor depletion following treatment with ligand and to determine if depletion is due to decreased expression of the Ahr gene that encodes the AHR. We found that depletion of AHR persisted for at least 72 hr after exposure to TCDD. Treatment with 3-methylcholanthrene caused a transient drop in total cell AHR, but the AHR levels returned to near pretreatment levels within 72 hr after the first exposure. TCDD treatment did not alter the levels of AHR mRNA as assessed by reverse transcription-polymerase chain reaction or slot blot assays. Thus, the decrease in AHR protein cannot be attributed to depression of transcription of the Ahr gene by TCDD. TCDD treatment did not alter the levels of the dimerization partner of the AHR, the AH receptor nuclear translocator protein (ARNT), or ARNT mRNA. In the presence of TCDD, both the AHR and the ARNT protein can be maintained at high levels in the nucleus if transcription is inhibited with actinomycin-D. In the absence of actinomycin-D, the AHR protein was lost rapidly, but the ARNT protein level in the cell was maintained. Together, these results suggest that the AHR protein is degraded through a selective mechanism that spares the ARNT protein and that the degradation pathway involves a protein that itself has a short half-life.

Binding of 2,3,7,8-tetrachlorodibenzo-p-dioxin to AH receptor in placentas from normal versus abnormal pregnancy outcomes.

Binding of [3H]2,3,7,8-tetrachlorodibenzo-p-dioxin to AH receptor was characterized in cytosol from human placentas in which the pregnancy outcome was normal compared with pregnancies in which there was some adverse outcome (premature birth; intrauterine growth retardation; structural abnormality). No significant difference was detected between normal and adverse outcomes in the concentration of AH receptor sites (Bmax) nor in the affinity with which [3H]TCDD bound to the receptor (Kd). Aryl hydrocarbon hydroxylase activity, a CYP1A1 enzyme regulated by the AH receptor, was elevated in placental microsomes from smokers; this elevation was associated with intrauterine growth retardation.

Characterization of polyclonal antibodies to the aromatic hydrocarbon receptor.

The aromatic hydrocarbon receptor (AHR) is a soluble intracellular protein that mediates most, if not all, the toxic effects of polycyclic aromatic hydrocarbons, such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and 3-methylcholanthrene. Initial binding of specific AHR ligands occurs in the cytoplasm; after a "transformation" step the ligand.receptor complex translocates to the cell nucleus and binds to specific DNA sequences, which act as transcriptional enhancers. We used a synthetic peptide--KLH conjugate corresponding to a 20 amino acid sequence at the N-terminal of the AHR to generate rabbit polyclonal anti-AHR antibodies. The antiserum was affinity purified, using the synthetic peptide conjugated to ovalbumin, and screened by western blot analyses, using [3H]TCDD photoaffinity labeled AHR. Specificity of the antiserum was confirmed by co-migration of photolabeled AHR with the major immunoreactive band identified by western blot. Further characterization showed that the antipeptide antibodies recognized equally both mouse and human AHR, which differ significantly in molecular mass (mouse Hepa-1 cells approximately 95 kDa; human LS180 cells approximately 110 kDa). The affinity-purified antibodies also recognized undenatured TCDD.AHR complexes, as determined by a shift in sedimentation of the [3H]TCDD.AHR complex on a sucrose gradient. The high specificity and sensitivity of this antibody were used to determine the fate of the AHR in cells exposed to [3H]TCDD. Western blot analysis revealed that TCDD exposure caused a dramatic decrease in total cellular AHR to about 20% pre-TCDD levels within 2 h after TCDD, which persisted up to 20 h after initial TCDD exposure. However, in the presence of actinomycin D or cycloheximide, nuclear AHR remained elevated in cells exposed to TCDD, at levels similar to or greater than the maximum previously observed after 1-h incubations. These data suggest that ligand-dependent downregulation of the AHR is the result of protein degradation by a short-lived protease.

Photoaffinity labeling of the Ah receptor with 3-[3H]methylcholanthrene and formation of a 165-kDa complex between the ligand-binding subunit and a novel cytosolic protein.

The aromatic hydrocarbon (Ah) receptor is a cytosolic protein that binds halogenated ligands such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and nonhalogenated ligands such as 3-methylcholanthrene (MC) and benzo[a]pyrene. The best characterized biological response mediated by the Ah receptor is induction of cytochrome P4501A1 (CYP1A1). Photoaffinity labeling of the Ah receptor has been reported only with halogenated ligands such as TCDD and some of its iodinated derivatives. In this study, photolabeling of the Ah receptor was achieved with the nonhalogenated aromatic hydrocarbon [3H]MC. Sources of Ah receptor were the mouse hepatoma cell line Hepa-1c1c9 and the human colon adenocarcinoma line LS180. Cytosolic fractions either were used in a crude form or were enriched by glycerol density gradient centrifugation. These then were incubated with [3H]MC, irradiated with UV light (> 300 nm), precipitated with acetone, and analyzed by SDS-polyacrylamide gel electrophoresis. The yield of photoadduct formation was lower with [3H]MC (approximately 1%) compared with [3H]TCDD (3.5%) in Hepa-1c1c9 cells. The same was true in LS180 cells, i.e. the yield was 0.2% for [3H]MC versus 5.48 +/- 0.26% for [3H]TCDD. The relative molecular mass of the [3H]MC-labeled receptor estimated by SDS-polyacrylamide gel electrophoresis was 94,600 +/- 2,400 (mean +/- S.E.) for Hepa-1c1c9 cells and 113,600 +/- 3,200 for LS180 cells; these are the same molecular masses as determined by photolabeling with [3H]TCDD. In velocity sedimentation assays of mouse cytosol, [3H]MC binds specifically to two cytosolic proteins: the 4 S carcinogen-binding protein and the Ah receptor (9 S). However, no photolabeling of the 4 S protein was detected in our experiments. [3H]MC photolabeling of the human Ah receptor from LS180 cells was detected only in experiments using enriched cytosolic preparations. In addition to the 95-kDa ligand-binding subunit, a specifically radiolabeled protein of 164,900 +/- 5,800 kDa was also detected in Hepa-1c1c9 cytosol photolabeled with [3H]MC, suggesting cross-linking, by MC, of another subunit of the multimeric Ah receptor complex to the ligand-binding subunit. Immunochemical analysis showed that the ligand-binding subunit of the Ah receptor is one component of the 165-kDa complex. The other protein in the complex could not be identified with antibodies to the heat shock proteins hsp90 or hsp70 or with antibodies to the p59 protein or Ah receptor nuclear translocator protein. The identity and function of the protein that becomes cross-linked to the ligand-binding subunit require further investigation.

In vitro transformation of the human Ah receptor and its binding to a dioxin response element.

Many biological effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD, dioxin) are mediated by a soluble intracellular protein, the Ah receptor (AhR). After binding of TCDD to the cytoplasmic AhR there occurs a poorly understood "transformation" step, wherein the TCDD-AhR complex is converted to a form that can bind to DNA with high affinity. The binding of transformed AhR to a specific dioxin-responsive element (DRE) upstream of a given gene stimulates transcriptional activation of that gene. Using a gel retardation assay we examined the interaction of transformed human cytosolic TCDD-AhR complexes with a synthetic DNA oligonucleotide containing a single DRE site. Transformation and DNA binding of human AhR in vitro was ligand dependent and specific for DRE-containing DNA. Unlike rodent hepatic AhR, in vitro transformation of human AhR was completely temperature dependent. Although at 4 degrees AhR binds ligand, no transformation of human TCDD-AhR complex was observed at 4 degrees even after 24 h; however, rapid transformation as measured by DNA binding was detectable as early as 10 min after warming to 22 degrees, with maximal binding by about 60 min. Calf thymus DNA-Sepharose or DRE-Sepharose column chromatography showed that transformed human cytosolic AhR interacts with DNA as a single species. The absolute temperature dependency of human AhR transformation mimics that observed in vivo and provides a useful system to study the mechanism of AhR transformation in detail.