David S. Riddick

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.

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.

NADPH-cytochrome P450 oxidoreductase: roles in physiology, pharmacology, and toxicology.

This is a report on a symposium sponsored by the American Society for Pharmacology and Experimental Therapeutics and held at the Experimental Biology 2012 meeting in San Diego, California, on April 25, 2012. The symposium speakers summarized and critically evaluated our current understanding of the physiologic, pharmacological, and toxicological roles of NADPH–cytochrome P450 oxidoreductase (POR), a flavoprotein involved in electron transfer to microsomal cytochromes P450 (P450), cytochrome b5, squalene mono-oxygenase, and heme oxygenase. Considerable insight has been derived from the development and characterization of mouse models with conditional Por deletion in particular tissues or partial suppression of POR expression in all tissues. Additional mouse models with global or conditional hepatic deletion of cytochrome b5 are helping to clarify the P450 isoform- and substrate-specific influences of cytochrome b5 on P450 electron transfer and catalytic function. This symposium also considered studies using siRNA to suppress POR expression in a hepatoma cell–culture model to explore the basis of the hepatic lipidosis phenotype observed in mice with conditional deletion of Por in liver. The symposium concluded with a strong translational perspective, relating the basic science of human POR structure and function to the impacts of POR genetic variation on human drug and steroid metabolism.

HIV protease inhibitors, saquinavir, indinavir and ritonavir: inhibition of CYP3A4-mediated metabolism of testosterone and benzoxazinorifamycin, KRM-1648, in human liver microsomes

The protease inhibitors, ritonavir, indinavir and saquinavir, the most potent anti-HIV drugs developed to date, interact with many drugs by competing for CYP3A4, an enzyme central to the metabolism of a wide variety of compounds. Human liver microsomes were used to compare inhibition by these three protease inhibitors. The inhibition was the greatest with ritonavir and indinavir and less potent with saquinavir.

Transcriptional suppression of cytochrome P450 2C11 gene expression by 3-methylcholanthrene.

Aromatic hydrocarbon receptor-mediated transcriptional up-regulation of cytochrome P450 (CYP) enzymes of the CYP1A subfamily by polycyclic aromatic hydrocarbons (PAHs) such as 3-methylcholanthrene (MC) is accompanied by down-regulation of rat hepatic CYP2C11 expression at the catalytic activity, protein, and mRNA levels. To gain insight into the molecular mechanism of this CYP2C11 suppression response, we have used a nuclear run-on assay to assess directly the effect of MC on the hepatic transcription rate of the CYP2C11 gene following in vivo administration of MC to adult male rats. A single intraperitoneal dose of MC (40 mg/kg) caused a 179-fold increase in the rate of CYP1A gene transcription at 6 hr, and the rate of CYP2C11 gene transcription was reduced by 51% at this time point, compared with vehicle controls. By 48 hr after MC treatment, the rates of CYP1A and CYP2C11 gene transcription were no longer significantly different from the corresponding vehicle controls. These results indicate for the first time that the suppression of hepatic CYP2C11 caused by in vivo administration of PAHs to adult male rats is at least partially due to a decrease in the rate of transcription of the CYP2C11 gene.

Role of the aromatic hydrocarbon receptor in the suppression of cytochrome P-450 2C11 by polycyclic aromatic hydrocarbons

The aromatic hydrocarbon (AH) receptor mediates the induction of cytochromes P-450 (CYP) of the CYP1A subfamily caused by polycyclic aromatic hydrocarbons (PAHs). CYP1A induction by PAHs is accompanied by down-regulation of CYP2C11, the predominant CYP expressed constitutively in the liver of male rats. We performed a structure-activity relationship study with a series of PAHs of the anthracene class in order to determine if the AH receptor is involved in CYP2C11 down-regulation. Anthracene, benz[a]anthracene, dibenz[a,c]anthracene, dibenz[a,h]anthracene, 7,12-dimethylbenz[a]anthracene, as well as 2,3,7,8-tetrachlorodibenzo-p-dioxin and 3-methylcholanthrene decreased CYP2C11 immunoreactive protein levels to varying degrees in primary rat hepatocytes cultured on a laminin-rich extracellular matrix. The binding affinity of the PAHs for the rat liver cytosolic AH receptor correlated with the potency for transforming the cytosolic AH receptor to its DNA-binding form. In addition, the ability of the PAHs to suppress CYP2C11 correlated with both the AH receptor binding affinity and the AH receptor transformation potency. These results suggest that the AH receptor plays a role in the down-regulation of CYP2C11 caused by PAHs.

Regulation of aryl hydrocarbon receptor expression and function by glucocorticoids in mouse hepatoma cells

The aryl hydrocarbon receptor (AHR) is a ligand-activated transcription factor that mediates most biological responses to 2,3, 7,8-tetrachlorodibenzo-p-dioxin (TCDD) and related aromatic hydrocarbons. Although the role of the AHR in control of drug metabolism and endocrine disruption is partly understood, we know little about the regulation of the AHR itself by endocrine factors. Our work with hypophysectomized rats suggested that hepatic AHR protein level is positively regulated by pituitary-dependent factors. A current hypothesis is that adrenal glucocorticoids elevate AHR expression and enhance responsiveness to AHR agonists. Dexamethasone (DEX) at concentrations that activate the glucocorticoid receptor (GR) increased AHR mRNA, protein, and TCDD-binding by approximately 50% in Hepa-1 mouse hepatoma cells. This response was blocked by the GR antagonist 17β-hydroxy-11β-[4-dimethylamino phenyl]-17α-[1-propynyl]estra-4,9-dien-3-one (RU486), suggesting GR involvement. This small magnitude increase in AHR levels was functionally significant; pretreatment of Hepa-1 cells with DEX caused a 75% increase in the maximum induction of an AHR-activated luciferase reporter plasmid by TCDD. A luciferase reporter under control of the proximal 2.5 kilobases of the mouse Ahr 5′-flanking region and promoter was induced approximately 2.5-fold by DEX when cotransfected with a mouse GR expression plasmid. This is the first demonstration that glucocorticoids increase AHR levels in hepatoma cells via a GR-dependent transcriptional mechanism, suggesting a novel aspect of cross-talk between the AHR and the GR.

Regulation of constitutive mouse hepatic cytochromes P450 and growth hormone signaling components by 3-methylcholanthrene.

3-Methylcholanthrene (MC) activates the aryl hydrocarbon receptor and increases expression of cytochrome P450 (P450) enzymes such as CYP1A1. MC also decreases expression of CYP2C11, the major hepatic P450 in male rats that is regulated by pulsatile growth hormone (GH) secretion via a pathway partially dependent on signal transducer and activator of transcription 5b (STAT5b). If disruption of this GH signaling pathway is important for MCs ability to suppress CYP2C11 transcription, we hypothesize that MC suppresses other male-specific genes (e.g., mouse Cyp2d9) regulated by pulsatile GH with STAT5b dependence. We examined the time course of MCs effects on hepatic P450s and GH signaling components in male C57BL/6 mice. P450 content, heme content, and NADPH P450 oxidoreductase activity were induced 2.3-, 1.8-, and 1.3-fold, respectively, by MC. MC dramatically induced CYP1A1 mRNA, protein, and catalytic activity. MC caused a 42% decrease in CYP2D9 protein, a 28% decrease in CYP2D9 mRNA, and a 27% decrease in testosterone 16α-hydroxylation activity. MC caused a pronounced decrease in CYP3A protein; however, there was no apparent change in testosterone 6β-hydroxylation activity, and changes in mRNA levels for CYP3A forms were relatively small. Expression of GH receptor and major urinary protein 2, a gene regulated by GH with STAT5b dependence, was decreased by MC at the mRNA level. These results show that MC suppresses mouse Cyp2d9, a pulsatile GH- and STAT5b-dependent male-specific gene, via a pretranslational mechanism that may involve disrupted GH signaling. Mouse CYP3A protein levels are dramatically decreased by MC via a mechanism that is not yet understood.

Aryl hydrocarbon receptor-dependence of dioxin's effects on constitutive mouse hepatic cytochromes P450 and growth hormone signaling components.

The aryl hydrocarbon receptor (AHR) has physiological roles in the absence of exposure to exogenous ligands, and mediates adaptive and toxic responses to the environmental pollutant 2,3,7,8-tetracholorodibenzo-p-dioxin (TCDD). A readily metabolized AHR agonist, 3-methylcholanthrene, disrupts the expression of mouse hepatic growth hormone (GH) signaling components and suppresses cytochrome P450 2D9 (Cyp2d9), a male-specific gene controlled by pulsatile GH via signal transducer and activator of transcription 5b (STAT5b). Using TCDD as an essentially nonmetabolized AHR agonist, and Ahr (-/-) mice as the preferred model to determine the AHR-dependence of biological responses, we now show that 2 mouse hepatic STAT5b target genes, Cyp2d9, and major urinary protein 2 (Mup2), are suppressed by TCDD in an AHR-dependent manner. TCDD also decreased hepatic mRNA levels for GH receptor, Janus kinase 2, and STAT5a/b with AHR-dependence. Without inducing selected hepatic inflammatory markers, TCDD caused AHR-dependent induction of Cyp1a1 and NADPH-cytochrome P450 oxidoreductase (Por) and suppression of Cyp3a11. In vehicle-treated mice, basal mRNA levels for CYP2D9, CYP3A11, POR, serum amyloid protein P, and MUP2 were influenced by Ahr genetic status. We conclude that AHR activation per se leads to dysregulation of hepatic GH signaling components and suppression of some, but not all, STAT5b target genes.

Induction of multidrug resistance transporter ABCG2 by prolactin in human breast cancer cells.

The multidrug transporter, breast cancer resistance protein, ABCG2, is up-regulated in certain chemoresistant cancer cells and in the mammary gland during lactation. We investigated the role of the lactogenic hormone prolactin (PRL) in the regulation of ABCG2. PRL dose-dependently induced ABCG2 expression in T-47D human breast cancer cells. This induction was significantly reduced by short-interfering RNA–mediated knockdown of Janus kinase 2 (JAK2). Knockdown or pharmacologic inhibition of the down-stream signal transducer and activator of transcription-5 (STAT5) also blunted the induction of ABCG2 by PRL, suggesting a role for the JAK2/STAT5 pathway in PRL-induced ABCG2 expression. Corroborating these findings, we observed PRL-stimulated STAT5 recruitment to a region containing a putative γ-interferon activation sequence (GAS) element at −434 base pairs upstream of the ABCG2 transcription start site. Introduction of a single mutation to the −434 GAS element significantly attenuated PRL-stimulated activity of a luciferase reporter driven by the ABCG2 gene promoter and 5′-flanking region containing the −434 GAS motif. In addition, this GAS element showed strong copy number dependency in its response to PRL treatment. Interestingly, inhibitors against the mitogen-activated protein kinase and phosphoinositide-3-kinase signaling pathways significantly decreased the induction of ABCG2 by PRL without altering STAT5 recruitment to the GAS element. We conclude that the JAK2/STAT5 pathway is required but not sufficient for the induction of ABCG2 by PRL.