Allan Okey Discusses Applying Microarray Techniques to Mechanistic Toxicology
Emerging Research Front Commentary, December 2010
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Article: Aryl hydrocarbon receptor regulates distinct dioxin-dependent and dioxin-independent gene batteries
Authors: Tijet, N;Boutros, PC;Moffat, ID;Okey,
AB;Tuomisto, J;Pohjanvirta, R |
Allan Okey talks with ScienceWatch.com and answers a few questions about this month's Emerging Research Front paper in the field of Pharmacoly & Toxicology.
Why do you think your paper is highly
cited?
Our Molecular Pharmacology paper constitutes the first study to apply a formal and systematic approach to identify the full spectrum of genes that are regulated by the aryl hydrocarbon receptor (AHR). Over the previous history of research on the AHR, beginning in 1976, only a few dozen genes had been shown to be under AHR control using conventional methods of biochemistry and molecular biology to study "one-gene-at-a-time."
Our research used gene expression arrays to interrogate a large fraction of the mouse transcriptome in a single experiment. Our paper is an early example of applying microarray techniques to mechanistic toxicology.
The most notable findings from our research using gene expression arrays were:
[1] The AHR is required for virtually all gene-expression responses to the dioxin, TCDD, in mouse liver. This is in keeping with previous studies (see below) which demonstrated that TCDD toxicity is negligible in mice in which the AHR has been knocked out and that toxicity is associated with transactivation of gene expression by the AHR. Our research confirms that without the AHR, the transcriptional response of genes to dioxin is essentially nil.
[2] Almost as many genes are affected by AHR-status alone as are affected by TCDD acting on the AHR. Products of the constitutively-responsive genes identified in our study may play important roles in normal physiology of the liver. Identifying physiologic roles for the AHR is the focus or recent research in several laboratories internationally.
[3] As had been observed in previous research, many genes were upregulated ("induced") by dioxin treatment. However, we also found that numerous genes are downregulated by TCDD when it acts on the AHR. Surprisingly, even more genes are suppressed simply by the AHR being present, independent of any exogenous AHR ligand. In past research on the AHR the emphasis has been on the receptor's ability to upregulate gene expression—for example, leading to induction of multiple cytochrome P450 enzymes that metabolize drugs and environmental chemicals.
Number of genes that respond to TCDD in mouse liver
versus rat liver. Animals were given a single injection of TCDD and gene
expression after 19 hours exposure was determined by microarray analysis.
As the intersection indicates, although at least 200 genes responded in
each species, only 33 genes responded in common in both species. (Modified
from: Boutros et al., BMC Genomics 9:419
doi:10.1186/1471-2164-9-419, 2008).
Our research shows, unexpectedly, that downregulation or suppression also is a prominent feature of transcriptional regulation by the AHR, although the biological and toxicological implications of downregulation remain murky as do the mechanisms by which downregulation occur.
Overall, our research reveals surprising diversity and breadth of AHR involvement in hepatic gene expression, both in response to dioxin and under basal conditions. The fact that the presence or absence of the AHR alters gene expression, even when animals are not treated with foreign chemicals, is in keeping with recent discoveries that several different endogenous substances can act as ligands for this receptor and potentially regulate fundamental biological processes.
Toxicity of potent environmental contaminants such as TCDD may result from their ability to disrupt normal signaling by endogenous AHR ligands or to over-stimulate pathways normally regulated by "normal" physiologic ligands.
Does it describe a new discovery, methodology, or
synthesis of knowledge?
Our paper does not describe new methodology. Rather, we combined gene expression-array technology with gene-knockout mice to determine the batteries of genes that the AHR regulates in mice treated with dioxin as well as in the absence of exogenous chemicals. Often remarkable new findings can be achieved in the frontier between different disciplines by combining methodologies of the two disciplines.
Would you summarize the significance of your paper
in layman's terms?
Dioxin-like chemicals are environmental contaminants that are considered "priority pollutants" and are of concern because they display extraordinarily potent toxic effects in several species of laboratory animal. The long-term goal of research in our laboratories is to understand the mechanisms by which dioxin-like chemicals cause toxicity and to attempt to use this understanding to better evaluate the risk to human health and health of the ecosystem posed by dioxins and related environmental chemicals.
The first step in toxicity of dioxins involves their binding to a specific protein, the aryl hydrocarbon receptor, which is found in virtually all cells of humans and other mammals as well as in birds, fish, and other vertebrate animals. Binding of dioxin to the receptor alters the activity of selected genes. This change in gene activity ultimately leads to toxic effects. The identities of the specific genes responsible for dioxin toxicity are not yet known.
The research reported in our paper attempts to first identify all of the genes that respond to dioxins in mouse liver. Then we conduct further experiments to determine which genes, out of all the genes that respond, are actually responsible for toxic effects.
How did you become involved in this research, and
how would you describe the particular challenges, setbacks, and
successes that you've encountered along the way?
The specific research reported in our Molecular Pharmacology paper derives from a long-standing and fruitful collaboration between Allan Okey's laboratory at the University of Toronto and the laboratories of Prof. Jouko Tuomisto (National Public Health Institute, Kuopio, Finland, presently called the Institute of Health and Welfare) and Prof. Raimo Pohjanvirta (now of the University of Helsinki, Finland).
Allan Okey's research since 1977 has focused on characterizing structure and function of the AHR (reviewed in his personal account of the development of the AHR research field in Toxicological Sciences 98:5-38, 2007). Jouko Tuomisto and Raimo Pohjanvirta brought their extensive expertise in dioxin toxicity to the collaboration. In the early 1980s they discovered a strain of rat that is remarkably resistant to lethal effects of the most potent environmental contaminant in the dioxin family, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD).
In our collaborative research we use the "resistant rat" model to reveal mechanisms and pathways that lead to major forms of dioxin toxicity. We use gene expression arrays to determine which specific genes respond differently to TCDD in genetically resistant rat strains than in standard laboratory rat strains that are highly susceptible to dioxin toxicity.
Our working hypothesis is that genes which respond differently to dioxins in sensitive versus resistant rat strains are prime candidates to be mechanistically involved in toxic outcomes. Our initial gene expression studies in the resistant rat model have been published: Franc et al., Arch. Toxicol. 82:809-30 (2008); Moffat et al., BMC Genomics 11:263 doi:10.1186/1471-2164-11-263 (2010).
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