The toxicogenomics of nuclear receptor agonists

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Abstract

Toxicogenomics is the study of the structure and output of the genome as it responds to adverse xenobiotic exposure. Large-scale transcriptional analysis, made possible through microarray technologies, enables us to study and understand the complexity of the biological effects of drugs and chemicals, with the ultimate goal of separating wanted effects from adverse effects. Nuclear receptors are attractive targets for drug discovery because, as ligand-activated transcription factors, they coordinately regulate the expression of at least hundreds of genes that, in turn, control much of cellular metabolism. Through toxicogenomics, it is becoming possible to understand the therapeutic effects of agonists within the context of toxic effects, classify new chemicals as to their complete effects on biological systems, and identify environmental factors that may influence safety or efficacy of new and existing drugs.

Introduction

The nuclear receptors comprise a large group of ligand-activated transcription factors that control much of cellular metabolism [1••]. Known endogenous ligands include a diversity of lipophilic compounds such as steroid hormones, fatty acids, eicosenoids, bile acids and oxysterols. Receptors for which endogenous ligands have not been identified (orphan receptors) can be activated by drugs and other xenobiotics. Because they regulate such a broad spectrum of metabolic responses, including glucose and lipid metabolism and inflammation, many nuclear receptors are attractive therapeutic targets for drug discovery. Established drug targets include the glucocorticoid receptor, estrogen receptor (ER), vitamin D receptor and the inappropriately named peroxisome proliferator activated receptors (PPAR). In addition to regulating normal metabolic processes, some nuclear receptors such as the pregnane X receptor (PXR) and constitutive androstane receptor (CAR) have evolved to regulate detoxification of xenobiotics through the induction of oxidative, conjugative and clearance pathways. In addition to drugs and endogenous ligands, nuclear receptors may also be inadvertent targets for environmental pollutants such as dioxin and pesticides.

As transcription factors, during the course of normal cell physiology each nuclear receptor type regulates a large number of genes that often overlap with those activated by other nuclear receptors. Also, although monomeric and homodimeric activation of transcription occurs for ER, progesterone and glucocorticoid receptors, many nuclear receptors form obligate heterodimers with the retinoid X receptor (RXR), hence much receptor crosstalk probably occurs. Beyond normal cell functional regulation, exposure to xenobiotics can take on an additional level of complexity. It is not uncommon for drugs and chemicals to act as agonists or antagonists for multiple receptors; for example, the hypolipemic drug bezafibrate is a peroxisome proliferating agent and a receptor pan-agonist with affinity for PPARα, γ and δ, and the antiprogestin mifepristone (RU486) is an antagonist for both the progesterone and glucocorticoid receptors. Further, during a toxic response to xenobiotics, nuclear-receptor-regulated transcriptional responses may become even more profound and incongruent as cells must simultaneously regulate both normal physiologic pathways and defense pathways. Finally, in addition to activation by ligands, many nuclear receptors are known to be further controlled by co-activators and repressors. Thus, understanding the complexity of nuclear-receptor-regulated transcriptional responses to toxic exposure is a daunting challenge that is becoming a major focus of toxicogenomics research.

Toxicogenomics is the study of the structure and output of the entire genome as it relates and responds to adverse xenobiotic exposure. Traditionally, the genes regulated by nuclear receptors in cells exposed to toxins have been explored at the mRNA and protein levels using northern and western blotting techniques. Though effective when studying the expression of individual genes, these approaches do not enable the understanding of the myriad of genes regulated by individual receptors or of the crosstalk between receptors. Discovery of the multiple genes regulated by each receptor type has thus been driven by technological advances in gene expressional analysis, most commonly including differential display, RT-PCR and DNA microarrays, and in the development of receptor transgenic and knockout animal models. Toxicogenomics is a new field, hence relatively few studies have been reported for key nuclear-receptor-regulated pathways involved in toxic responses. The nuclear receptors currently recognized and studied as important to toxicology (Figure 1) include those that are involved in detoxification and drug interactions (PXR and CAR), known or potential drug targets for metabolic diseases (PPARα and γ; farnesoid X receptor [FXR]; liver X receptors [LXR]), targets for man-made environmental toxins (ER; the thyroid hormone receptor [TR]; and the aryl hydrocarbon receptor [AhR]), and the RXR, which can act as a homodimer but also forms a heterodimer with many other receptors (including PXR, CAR, PPAR, FXR, LXR and TR). Comprehensive reviews on the regulation of gene expression by the LXR and FXR nuclear receptors have been recently published 2., 3., hence these receptors are not further discussed here.

Section snippets

PXR and CAR

The orphan receptors PXR and CAR [4] are transcriptional regulators of many genes involved in drug metabolism and drug transport. Of particular focus is the induction of the phase I enzymes including the cytochrome p-450 (CYP) monoxygenases CYP3A and CYP2B. CYP3A is responsible for the oxidative metabolism of most pharmaceuticals along with many dietary and environmental xenobiotics and is particular concern for drug–drug and drug–food interactions. In addition to phase I oxidative enzymes, PXR

PPARα, PPARγ and RXR

The family of receptors collectively known as the peroxisome proliferators activated receptors has three distinct isotypes: PPARα, PPARβ (also called PPARδ) and PPARγ. Collectively they are known to function as regulators of glucose homeostasis, lipid metabolism, cell proliferation and cell differentiation. The toxic effects of PPARγ agonists have recently been reviewed [20], but no toxicogenomics study has been reported.

To date, only toxicogenomics studies on PPARα agonists have been

AhR

AhR is structurally distinct from the nuclear receptor superfamily and is a member of the basic helix–loop–helix-Per-ARNT-Sim homology protein superfamily. Given its functional similarity to the steroid hormone nuclear receptor family and its importance in toxicology, it is often considered along with other nuclear receptors and hence is discussed here. Upon ligand binding, AhR forms a heterodimer with its partner Arnt, and the AhR–Arnt complex binds to the xenobiotic responsive element to

ER and TR

Both ER and TR have been shown to be targets for environmental toxins. The 2,2-bis(p-hydroxyphenyl)-1,1,1-trichloroethane (HPTE) metabolite of the organochlorine pesticide methoxychlor is an ERα-selective agonist with ERβ and androgen receptor antagonist activities. In studies comparing HPTE to 17 β-estradiol and the antiandrogen flutamide, Waters et al. [34] found approximately 50 genes in ovary and uterus to be differentially regulated out of 728 genes on two different cDNA arrays. These

Conclusions

As transcription factors regulating the expression of hundreds of genes, the nuclear receptors are obvious candidates for studies using microarrays and similar technologies to understand the comprehensive influence of agonists, both on- and off-target, on gene expression. With time, it should be possible to understand the downstream biological effects controlled by each receptor individually and, through use of carefully constructed knockout and transgenic animals along with selective agonists,

References and recommended reading

Papers of particular interest, published within the annual period of review, have been highlighted as:

  • of special interest

  • ••

    of outstanding interest

Acknowledgements

The author acknowledges the excellent assistance of Marta Restrepo in the preparation of this manuscript.

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