Human aryl hydrocarbon receptor polymorphisms that result in loss of CYP1A1 induction.

The aryl hydrocarbon receptor (AHR) binds xenobiotic chemicals such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and regulates transcription of the P4501 subfamily that metabolizes many carcinogens. In humans, the most frequent polymorphism is R554L. We report here an additional two polymorphisms in AHR that show apparent linkage disequilibrium with the codon 554 polymorphism: the first is a previously described polymorphism, V570I; the second is a novel human AHR polymorphism, P571S. In vitro expression of these variant forms showed normal ligand binding and DNA binding activities. However, transient expression experiments revealed that the combined Ile(570) + Lys(554) variant failed to support TCDD-dependent induction of CYP1A1 expression. It is possible that the abrogation of CYP1A1 induction in the combined Lys(554) + Ile(570) variant may reduce susceptibility of the host to the carcinogenic effects of polycyclic aromatic hydrocarbons. This combination of variant genotypes is rare and appears to be confined primarily to persons of African descent.

Persistent, low-dose 2,3,7,8-tetrachlorodibenzo-p-dioxin exposure: effect on aryl hydrocarbon receptor expression in a dioxin-resistance model.

Most toxic effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) are mediated by the aryl hydrocarbon receptor (AHR). A single, acute dose of TCDD can alter its own receptor levels thus complicating evaluation of dose-response relationships for AHR-mediated events. Since environmental exposure to dioxins is typically of a repeated low-dose nature, we examined the effect of such exposure on AHR expression. Three rat strains differing greatly in their sensitivity to acute TCDD lethality, Long-Evans (Turku AB) (L-E) (LD50 approximately 10 microg/kg); Sprague Dawley (SD) (LD50 approximately 50 microg/kg); and Han/Wistar (Kuopio) (H/W) (LD50 > 9600 microg/kg), were administered TCDD intragastrically, biweekly for 22 weeks producing doses equivalent to 0, 10, 30, and 100 ng/kg/day. Changes in hepatic AHR levels were quantitated at the protein level by radioligand binding and immunoblotting and at the mRNA level by RT-PCR. Cytosolic AHR protein was elevated at 10 or 30 ng/kg/day TCDD in SD and L-E rats; AHR mRNA was also elevated at these doses, suggesting a pretranslational mechanism. There was no apparent relationship between TCDD-induced AHR regulation and strain sensitivity to TCDD. Overall, "subchronic" TCDD did not greatly perturb AHR expression. The maintenance of relatively constant receptor levels in the face of persistent agonist stimulation is in contrast to the sustained depletion of AHR by TCDD observed in cell culture and to the fluctuations in AHR observed hours to days following acute TCDD exposure in vivo. Changes in AHR levels may affect dose-response relationships; the effect of TCDD on its own receptor at environmentally relevant dosing schemes is therefore important to risk assessment.

Ethnic variability in the allelic distribution of human aryl hydrocarbon receptor codon 554 and assessment of variant receptor function in vitro.

The aryl hydrocarbon receptor (AHR) is a ligand-dependent transcriptional regulator of several genes including the cytochrome P4501 (CYP1) family as well as genes encoding factors involved in cell growth and differentiation. In mice, several polymorphic forms of the AHR are known, some of which have altered affinity for toxic and carcinogenic ligands. Remarkably little genetic variation has been detected in the human AHR gene. In studies on human AHR, Kawajiri et al. (Pharmacogenetics 1995; 5:151-158) reported a variation at codon 554 that results in an amino acid change from arginine to lysine; the frequency of the variant allele in a Japanese population (n = 277) was 0.43. We investigated the Lys554 allele in 386 individuals of various ethnic origins and found the frequency to be: 0.58 in Ivory Coast Africans (n = 58); 0.53 in a mixed African group (n = 20); 0.39 in Caribbean-Africans (n = 55); 0.32 in Canadian Chinese (n = 41); 0.14 in North American Indians (n = 47); 0.12 in French Canadian Caucasians (n = 20); 0.11 in a mixed ethnicity North American group (n = 45); 0.09 in Canadian Inuits (n = 22); and 0.07 in German Caucasians (n = 78). We expressed the human Lys554 allele in an in-vitro transcription-translation system and found that the receptor bearing the R554L substitution had an equivalent ability to that of the wild-type receptor to bind to a dioxin-responsive element following treatment with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). The Lys554 allele also was equivalent to the wild-type receptor at stimulating CYP1A1 mRNA expression when transfected into TCDD-treated receptor-deficient mouse Hepa-1 cells. It is not yet known if any of the wide variations in allele frequency at codon 554 are related to ethnic differences in susceptibility to adverse effects of environmental chemicals.

In vivo up-regulation of aryl hydrocarbon receptor expression by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in a dioxin-resistant rat model.

The aryl hydrocarbon receptor (AHR) mediates toxicity of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and regulates expression of several genes such as CYP1A1. Little is known about what regulates expression of the AHR itself. We tested the ability of TCDD to alter in vivo expression of its own receptor in rat strains that are susceptible to TCDD lethality [Long-Evans (Turku AB) (L-E) and Sprague Dawley (SD)] and in a rat strain that is remarkably resistant to TCDD lethality [Han/Wistar (Kuopio) (H/W)]. Rats were administered a single, intragastric dose of 5 or 50 microg/kg of TCDD. Hepatic cytosol, nuclear extract, and RNA were prepared at 1, 4, and 10 days after TCDD exposure. AHR expression was assessed at three levels: ligand binding function, immunoreactive protein and mRNA. TCDD at 5 microg/kg produced a 2- to 3-fold increase in cytosolic AHR in all strains; 50 microg/kg produced depletion at day 1 followed by recovery in SD and H/W but not L-E rats. Both the increase in AHR above basal levels and the recovery from initial depletion were accompanied by elevations in steady-state AHR mRNA, suggesting a pre-translational mechanism for AHR regulation by its own ligand. This up-regulation in vivo is in contrast to the sustained depletion of AHR caused by TCDD in cell culture. There was no clear relationship between AHR regulation and strain sensitivity; thus, the large inherent strain differences in susceptibility to TCDD lethality probably are not explained by differential regulation of AHR by TCDD.

Treatment-resistance to clozapine in association with ultrarapid CYP1A2 activity and the C-->A polymorphism in intron 1 of the CYP1A2 gene: effect of grapefruit juice and low-dose fluvoxamine.

Antipsychotic response to clozapine varies markedly among patients with schizophrenia. The disposition of clozapine is dependent, in part, on the cytochrome P-450 (CYP) 1A2 enzyme in vivo. In theory, a very high CYP1A2 activity may lead to subtherapeutic concentrations and treatment resistance to clozapine. This prospective case study evaluates the clinical significance of ultrarapid CYP1A2 activity and a recently discovered single nucleotide (C --> A) polymorphism in intron 1 of the CYP1A2 gene (CYP1A2*F) for treatment resistance to clozapine. In addition, we describe the effect of grapefruit juice or low-dose fluvoxamine (25-50 mg/d) coadministration on clozapine and active metabolite norclozapine steady-state plasma concentration and antipsychotic response.

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.

Regulation of cytochrome P450 enzymes by aryl hydrocarbon receptor in human cells: CYP1A2 expression in the LS180 colon carcinoma cell line after treatment with 2,3,7,8-tetrachlorodibenzo-p-dioxin or 3-methylcholanthrene.

It has been difficult to study the regulation of cytochrome P4501A2 (CYP1A2) because expression of this enzyme is reported to be limited or absent in cell culture. We found that CYP1A2 can be induced significantly by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), 3-methylcholanthrene (MC), or benz[a]anthracene in the human colon carcinoma cell line LS180. TCDD and MC each caused a dramatic elevation of CYP1A2 mRNA, as assessed by reverse transcription-polymerase chain reaction or by northern blot analysis. TCDD also increased immunoreactive CYP1A2 protein and the activity of phenacetin-O-deethylase, a diagnostic catalytic marker for CYP1A2. The induction of CYP1A2 at all levels (mRNA, protein, catalytic activity) was concentration- and time-dependent: the EC50 for mRNA induction by TCDD = 0.5 nM, and by MC = 1.4 microM. Inducible CYP1A2 mRNA also was detected at lower levels in two other human cell lines, the hepatoma cell line HepG2 and the breast carcinoma cell line MCF-7. CYP1A1 and CYP1B1, additional CYP1 enzymes regulated by the aryl hydrocarbon receptor (AHR), also were inducible by TCDD and MC in LS180 cells; their concentration-dependent induction was highly correlated with induction of CYP1A2 at mRNA, protein, and catalytic levels. CYP1B1 was constitutively expressed and inducible in the LS180, MCF-7, and HepG2 cell lines as well as in the human choriocarcinoma cell line JEG-3 and the squamous cell carcinoma line A431. CYP1A2 was neither constitutively expressed nor inducible in A431 or JEG-3 cells. The expression of mRNAs encoding the regulators of CYP1 enzymes-the AHR and its heterodimerization partner, the ARNT (AH receptor nuclear translocator) protein-was not altered by treatment with TCDD or MC. However, the cytosolic content of AHR protein and ARNT protein was depleted substantially following treatment with TCDD. The LS180 cell line should constitute a good model for further mechanistic studies on AHR-regulated CYP1A2 expression.

Point mutation in intron sequence causes altered carboxyl-terminal structure in the aryl hydrocarbon receptor of the most 2,3,7,8-tetrachlorodibenzo-p-dioxin-resistant rat strain.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is the most potent dioxin. There are exceptionally wide inter- and intraspecies differences in sensitivity to TCDD toxicity with Han/Wistar (H/W) (Kuopio) rats being the most resistant mammals tested. A peculiar feature of H/W rats is that despite their unresponsiveness to the acute lethality of TCDD, their sensitivity to other biological impacts of TCDD (e.g., CYP1A1 induction) is preserved. The biological effects of TCDD are mediated by the aryl hydrocarbon receptor (AhR). We recently found that the AhR of H/W rats (about 98 kDa) is smaller than the receptor in other rat strains (106 kDa). In the present study, molecular cloning and sequencing of the H/W rat AhR revealed that the reason for its smaller size is a deletion/insertion-type change at the 3' end of exon 10 in the receptor cDNA. This change emanates from a single point mutation at the first nucleotide of intron 10, resulting in altered mRNA splicing. At the protein level, the mutation leads to a total loss of either 43 or 38 amino acids (with altered sequence for the last seven amino acids in the latter case) toward the carboxyl-terminal end in the trans-activation domain of the AhR. H/W rats also harbor a point mutation in exon 10 that will cause a Val-to-Ala substitution in codon 497, but this occurs in a variable region of the AhR. These findings suggest that there is a relatively small region in the AhR trans-activation domain that may be capable of providing selectivity to its function.

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.

Aromatic hydrocarbon receptor in cultured fetal cells from C57BL/6J and DBA/2J mice: similarity in molecular mass to receptors in adult livers.

In liver of adult responsive C57BL/6J (B6) mice the aromatic hydrocarbon receptor (AHR) has high affinity for specific halogenated aromatic hydrocarbons, such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), as well as nonhalogenated aromatic hydrocarbons (PAHs), such as benz[a]anthracene (BA) or 3-methylcholanthrene (MC). In livers of adult nonresponsive DBA/2J (D2) mice TCDD binds to a low-affinity variant form of AHR. Both TCDD and MC induce aryl hydrocarbon hydroxylase (AHH) in adult B6 mice, whereas adult D2 mouse liver is nonresponsive to MC. In fetal cell cultures derived from D2 mice AHH is induced by PAHs such as MC or BA, and these PAHs bind to cytosolic AHR (P.A. Harper, C.L. Golas, and A.B. Okey. Mol. Pharmacol. 40: 818-826, 1991). We compared AHR from fetal cell cultures with AHR from adult livers to determine whether there was some structural differences in receptors expressed in fetal cell culture that might permit cells from "nonresponsive" mice to respond to PAHs. The apparent molecular mass of AHR from cells cultured from 18-day fetuses is identical with that from adult liver within each strain of inbred mice tested (M(r) approximately 95 kDa in B6 and approximately 105 kDa in D2 mice). The AHR in D2 fetal cells was able to activate a transfected chloramphenicol acetyltransferase linked to a dioxin-responsive element nucleotide sequence (DRE-CAT) when the cells were treated with TCDD or MC. The potency of CAT expression in D2 fetal cells was similar to that in B6 fetal cells. Our data suggest that the responsiveness of fetal cells from "nonresponsive" mice is likely mediated by AHR in these cells but is not due to expression of a different allelic form of AHR ligand-binding subunit in fetal cells versus adult liver.

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.

Molecular biology of the aromatic hydrocarbon (dioxin) receptor.

The aromatic hydrocarbon (AH) (dioxin) receptor was discovered almost 20 years ago and achieved notoriety as the front-line site of action of highly toxic environmental chemicals such as halogenated dioxins and polychlorinated biphenyls. Increasing evidence suggests that the AH receptor plays a key role in proliferation and differentiation of cells exposed to dioxins and, perhaps, to endogenous ligands. Recent cloning of the AH receptor and its indispensable partner, the AH-receptor-nuclear-translocator protein, has opened new opportunities to determine how the AH receptor functions, how it evolved and what its multiple roles might be in normal physiology as well as in toxicology. This review by Allan Okey, David Riddick and Patricia Harper aims to provide a brief history of AH receptor research and gives a timely summary of what is known and what is not known about the structure and function of this fascinating protein.

2,3,7,8-Tetrachlorodibenzo-p-dioxin versus 3-methylcholanthrene: comparative studies of Ah receptor binding, transformation, and induction of CYP1A1.

Halogenated aromatic hydrocarbons such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and polycyclic aromatic hydrocarbons such as 3-methylcholanthrene (MC) cause transcriptional activation of the CYP1A1 gene via their interaction with the aromatic hydrocarbon (Ah) receptor. Direct radioligand binding and competitive binding studies demonstrated that the cytosolic Ah receptor from the mouse hepatoma cell line Hepa-1 bound TCDD with an affinity approximately 3-4-fold greater than that for MC. However, TCDD was approximately 1,000-fold more potent than MC as an inducer of CYP1A1-mediated aryl hydrocarbon hydroxylase activity in cultured Hepa-1 cells as assessed at 14 h following exposure to inducer. To understand the basis for this quantitative discrepancy between Ah receptor binding affinity and CYP1A1 induction potency, we systematically compared TCDD and MC for their abilities to activate sequential events in the CYP1A1 induction mechanism that occur subsequent to initial binding to the cytosolic Ah receptor. Using a gel retardation assay, TCDD and MC were shown to be equipotent in causing in vitro transformation of the cytosolic Ah receptor to its DNA-binding form. In addition, the transformed Ah receptor bound to a specific dioxin-responsive enhancer sequence with the same apparent affinity when MC was the ligand as when TCDD was the ligand. At an early time point (i.e. 2 h) in the CYP1A1 induction process, TCDD was only approximately 4-25-fold more potent than MC in stimulating the nuclear uptake of the ligand-Ah receptor complex, and the two ligands displayed a relatively small difference (> or = 10-fold) in CYP1A1 mRNA induction potency. When assessed at 4 h following ligand treatment, TCDD was only approximately 10-fold more potent than MC as an aryl hydrocarbon hydroxylase inducer, suggesting a time-dependent reduction in the potency of MC in intact cells. Exposure of Hepa-1 cells to MC over a 16-h time course resulted in an increased ability of these cells to convert [3H]MC to alkali-extractable metabolites. Our data are consistent with the idea that TCDD and MC display relatively small differences in their intrinsic abilities to activate Ah receptor-mediated events. The reduced biological potency of MC observed in intact cells and whole animals is at least partially due to the more rapid metabolic inactivation of this ligand compared with the poorly metabolized TCDD. By extension, the extraordinary toxicity of TCDD may not be explained solely by its high affinity for the cytosolic Ah receptor.

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.

The Ah receptor: mediator of the toxicity of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and related compounds.

A considerable body of research over the past fifteen years establishes that in laboratory animals the Ah (aromatic hydrocarbon) receptor (AhR) mediates most, if not all, toxic effects of halogenated aromatic hydrocarbons such as polychlorinated dibenzo-p-dioxins, polychlorinated dibenzofurans, and polyhalogenated biphenyls. More recently the AhR has been shown to also exist in a wide variety of human tissues and human cell lines. In general the AhR in humans appears to function very much like the AhR in rodents. However, the affinity with which toxic HAHs such as 2,3,7,8-tetrachlorodibenzo-p-dioxin bind to the AhR from human sources generally is lower than the affinity with which these HAHs bind to the Ah receptors from rodent tissues. This lower affinity may explain, in part, why the human species seems less sensitive than many laboratory animals to the effects of HAHs. The AhR enhances transcription of genes encoding cytochrome P450 enzymes in the CYP1A subfamily, but most of the toxic effects of HAHs do not seem to require P450 induction per se. Recent molecular approaches to the mechanism of HAH toxicity indicate that the AhR also may mediate expression of several other genes, including genes that regulate cell growth and differentiation. Despite the expanding repertoire of cellular responses known to be altered by HAHs (potentially through the AhR) it is not yet clear which AhR-mediated actions are the key events in HAH toxicity. Within the past year two subunits of the AhR have been cloned; this cloning, along with other molecular investigations, should greatly expand our opportunity to understand the specific mechanisms and pathways by which HAHs cause toxicity.

Ah receptor binding properties of indole carbinols and induction of hepatic estradiol hydroxylation.

The effect of route of administration on the ability of indole-3-carbinol (13C), an anticarcinogen present in cruciferous vegetables, to induce estradiol 2-hydroxylase (EH) in female rat liver microsomes was investigated and compared to that of its main gastric conversion product, 3,3'-diindolylmethane (DIM). This dimer was more potent than 13C after either oral or intraperitoneal administration and was also a better in vitro inhibitor of EH in control and 13C-induced hepatic microsomes. The induction of both CYP1A1 and 1A2 in about equal amounts by 13C and DIM as well as of CYP2B1/2 was demonstrated using monoclonal antibodies. DIM, isosafrole, beta-naphthoflavone, 3-methylcholanthrene and naringenin added in vitro inhibited EH strongly in induced microsomes but gestodene was a better inhibitor of estrogen 2-hydroxylation in liver microsomes from untreated female rats. The binding affinities of 13C and DIM to the Ah receptor were compared to that of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) by competition studies, and the IC50 values were shown to be 2.0 x 10(-9) M, 5.0 x 10(-5) M and 2.3 x 10(-3) M for TCDD, DIM and 13C, respectively. The ability of 13C or DIM to cause in vitro transformation of the Ah receptor to a form able to bind to the dioxin-responsive element-3 (DRE3) was compared to that of TCDD and shown to parallel their abilities to compete for binding of [3H]TCDD to the Ah receptor. These experiments confirm and extend the proposals that dietary indoles induce specific cytochrome P450s in rat liver by a mechanism possibly involving the Ah receptor. The induced monooxygenases, in turn, increase the synthesis of 2-hydroxylated estrogens in the competing pathways of 2- and 16 alpha-hydroxylation which decreases the levels of 16 alpha-hydroxyestrone able to form stable covalent adducts with proteins including the estrogen receptor. Such steroid-protein interaction has been correlated with mammary carcinogenesis.