Allan B. Okey

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.

Aryl Hydrocarbon Receptor Regulates Distinct Dioxin-Dependent and Dioxin-Independent Gene Batteries

Conventional biochemical and molecular techniques identified previously several genes whose expression is regulated by the aryl hydrocarbon receptor (AHR). We sought to map the complete spectrum of AHR-dependent genes in male adult liver using expression arrays to contrast mRNA profiles in Ahr-null mice (Ahr–/–) with those in mice with wild-type AHR (Ahr+/+). Transcript profiles were determined both in untreated mice and in mice treated 19 h earlier with 1000 μg/kg 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Expression of 456 ProbeSets was significantly altered by TCDD in an AHR-dependent manner, including members of the classic AHRE-I gene battery, such as Cyp1a1, Cyp1a2, Cyp1b1, and Nqo1. In the absence of exogenous ligand, AHR status alone affected expression of 392 ProbeSets, suggesting that the AHR has multiple functions in normal physiology. In Ahr–/– mice, only 32 ProbeSets exhibited responses to TCDD, indicating that the AHR is required for virtually all transcriptional responses to dioxin exposure in liver. The flavin-containing monooxygenases, Fmo2 and Fmo3, considered previously to be uninducible, were highly induced by TCDD in an AHR-dependent manner. The estrogen receptor α as well as two estrogen-receptor-related genes (α and γ) exhibit AHR-dependent expression, thereby extending cross-talk opportunities between the intensively studied AHR and estrogen receptor pathways. p53 binding sites are over-represented in genes down-regulated by TCDD, suggesting that TCDD inhibits p53 transcriptional activity. Overall, our study identifies a wide range of genes that depend on the AHR, either for constitutive expression or for response to TCDD.

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.

Polymorphisms in the human AH receptor.

The AH receptor (AHR) mediates toxicity of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) as well as induction of three cytochrome P450 enzymes and certain Phase II enzymes. In laboratory animals, genetic variations in the AHR lead to substantial differences in sensitivity to biochemical and toxic effects of TCDD and related compounds. Relatively few polymorphisms have been discovered in the human AHR gene; these occur predominantly in exon 10, a region that encodes a major portion of the transactivation domain of the receptor that is responsible for regulating expression of other genes. In human populations there is a wide range of variation in responses regulated by the AHR for example, induction of CYP1A1. Some variation in human responsiveness likely is due to genetically based variations in AHR structure. Thus far, however, only one pair of polymorphisms, those at codons 517 and 570, has been shown to have a clear cut and strong effect on the phenotype of an AHR-mediated response. The search continues for polymorphisms that alter AHR function because this receptor is a central factor in determining responses to important environmental contaminants and also plays a physiologic role in early development in mammals.

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.

Red meat intake, doneness, polymorphisms in genes that encode carcinogen-metabolizing enzymes and colorectal cancer risk

Colorectal cancer literature regarding the interaction between polymorphisms in carcinogen-metabolizing enzymes and red meat intake/doneness is inconsistent. A case-control study was conducted to evaluate the interaction between red meat consumption, doneness, and polymorphisms in carcinogen-metabolizing enzymes. Colorectal cancer cases diagnosed 1997 to 2000, ages 20 to 74 years, were identified through the population-based Ontario Cancer Registry and recruited by the Ontario Family Colorectal Cancer Registry. Controls were sex-matched and age group-matched random sample of Ontario population. Epidemiologic and food questionnaires were completed by 1,095 cases and 1,890 controls; blood was provided by 842 and 1,251, respectively. Multivariate logistic regression was used to obtain adjusted odds ratio (OR) estimates. Increased red meat intake was associated with increased colorectal cancer risk [OR (>5 versus ≤2 servings/wk), 1.67 (1.36-2.05)]. Colorectal cancer risk also increased significantly with well-done meat intake [OR (>2 servings/wk well-done versus ≤2 servings/wk rare-regular), 1.57 (1.27-1.93)]. We evaluated interactions between genetic variants in 15 enzymes involved in the metabolism of carcinogens in overcooked meat (cytochrome P450, glutathione S-transferase, UDP-glucuronosyltransferases, SULT, NAT, mEH, and AHR). CYP2C9 and NAT2 variants were associated with colorectal cancer risk. Red meat intake was associated with increased colorectal cancer risk regardless of genotypes; however, CYP1B1 combined variant and SULT1A1-638G>A variant significantly modified the association between red meat doneness intake and colorectal cancer risk. In conclusion, well-done red meat intake was associated with an increased risk of colorectal cancer regardless of carcinogen-metabolizing genotype, although our data suggest that persons with CYP1B1 and SULT1A1 variants had the highest colorectal cancer risk. (Cancer Epidemiol Biomarkers Prev 2008;17(11):3098–107)

Detection and characterization of [3H]2,3,7,8-tetrachlorodibenzo-p-dioxin binding to Ah receptor in a rainbow trout hepatoma cell line

Ah receptor was identified and characterized in cytosol and nuclear extracts from the rainbow trout hepatoma cell line RTH-149. The cytosolic receptor was detectable with both halogenated ([3H]2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)) and non-halogenated ([3H]3-methylcholanthrene and [3H]benzo[a]pyrene) aromatic hydrocarbons and sedimented at approximately 9 S after velocity sedimentation on sucrose gradients. The apparent binding affinity (kd) of cytosolic Ah receptor was always less than 1 nM as derived from Scatchard or Woolf plot analyses. The same analyses indicated a concentration of Ah receptor in the RTH-149 cells of approximately 20 fmol/mg cytosolic protein or approximately 4400 receptor sites per cell. Thus, this trout hepatoma cell line has a low concentration of high-affinity binding sites in comparison to Ah receptor concentrations in cytosol obtained from rodent tissues. Incubation of whole cells with the radioligand [3H]TCDD resulted in transformation of the cytosolic Ah receptor to a nuclear binding form which could be detected as a specifically labeled peak sedimenting at approximately 6 S on sucrose gradients. Aryl hydrocarbon hydroxylase was induced after exposure of RTH-149 cells to TCDD or benz[a]anthracene for 24 hr in culture. These data demonstrate the existence of the Ah receptor in a cell line derived from a nonmammalian species and provide an additional step toward understanding the mechanisms by which fish respond to specific aquatic contaminants.

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.

Advances in analytical techniques for polychlorinated dibenzo-p-dioxins, polychlorinated dibenzofurans and dioxin-like PCBs

Analytical techniques for the determination of polychorinated dibenzo-p-dioxins (PCDD), polychlorinated dibenzofurans (PCDF) and dioxin-like PCBs (DLPCB) are reviewed. The focus of the review is on recent advances in methodology and analytical procedures. The paper also reviews toxicology, the development of toxic equivalent factors (TEF) and the determination of toxic equivalent quantity (TEQ) values. Sources, occurrence and temporal trends of PCDD/PCDF are summarized to provide examples of levels and concentration ranges for the methods and techniques reviewed.

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.