Unraveling the Complexities of the Mechanism of Action of Dioxins
National Institute of Environmental Health Sciences, National Institutes of Health, PO Box 12233, MD EC-34, 111 T. W. Alexander Drive, Research Triangle Park, North Carolina 27709
For correspondence via fax: (301) 451-5596. E-mail: walker3{at}niehs.nih.gov.
Received November 15, 2006; accepted November 15, 2006
Devoutly to be wish'd. To die, to sleep;To sleep: perchance to dream: ay, there's the rub:
For in that sleep of death what dreams may come,
When we have shuffled off this mortal coil,
Must give us pause: there's the respect
That makes calamity of so long life;
William Shakespeare (From Hamlet)
For some, the continuing saga of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and the aryl hydrocarbon receptor (AhR) are like one of those daytime soap operas that are long past their prime and where there are no new stories to be told. For others, it is a constant source of new twists and turns, where the actors come and go on a regular basis and where, no matter how many times you watch, there are still cliffhangers to keep you watching again tomorrow. This is because no matter whether its General Hospital or Hamlet, understanding what may seem to be a simple story is never quite so simple.
The story of TCDD and the AhR is over 30 years in the telling now. From the identification of TCDD as one of the most potent toxicants and carcinogens known to toxicologists, through the identification and dissection of the Ah receptor– and Ah receptor nuclear translocator (ARNT) protein–signaling pathways, to the more recent understanding of the key roles of the AhR in development (Schmidt and Bradfield, 1996
; Teeguarden and Walker, 2003). So the story goes on. Why? Because despite these decades of research, we still do not have a clear mechanism of action leading from ligand binding of TCDD or related ligand to the AhR and the ultimate development of toxicities. We have modes of action and we have hypotheses. And "ay there's the rub," for therein lies a lot of the uncertainty considered by some of the health risk posed by TCDD to human health. We know TCDD is a potent hepatotoxicant and carcinogen in rodents, but other than binding to the AhR, we are still not sure why, or how. Consequently, there remains uncertainty about how the findings in rodent models apply to humans, particularly at the low-exposure levels encountered by the general population. In particular, the low-dose cancer risk and whether the shape of the dose-response is linear or nonlinear or exhibits a "threshold" continue to be a source of debate. For many, TCDD and AhR has become one of the models de rigueur for dealing the complexity of nongenotoxic carcinogens, particularly as it applies to risk assessment.
One of the key steps to understanding the low-dose risk and the extrapolation across species is the knowledge of the mechanisms of development of toxicity both within and across species, within the context of how dose, duration of exposure, and life stage affect these mechanisms. Twenty-five years ago, Poland and Knutson proposed a general model for toxicity of TCDD whereby tissues exhibiting "toxicity" express a ligand-activated receptor–mediated pleiotropic response of a battery of "restricted" genes in addition to the "limited" pleiotropic response, characterized by induction of a drug-metabolizing enzyme battery (Poland and Knutson, 1982). Not surprisingly, the advent of new technologies has been applied over the years to the TCDD/AhR and the examination of this model. From the first use of differential cDNA hybridization for the identification of dioxin responsive genes, CYP1B1, PAI-2, and IL-1ß (Sutter et al., 1991, 1994), to the use of microarrays (Frueh et al., 2001; Martinez et al., 2002; Puga et al., 2000), the identification of the biological targets and pathways that comprise these pleiotropic responses in tissues following ligand binding to the AhR has been a key goal.
Two recent papers published in the December issue of Toxicological Sciences (Boverhof et al., 2006; Ovando et al., 2006) attempt to shed light on this by using different yet complementary comparative analyses of the genomic transcriptional response to TCDD and related AhR receptor ligands. Together these provide the most comprehensive evaluation to date of the time-, dose-, and species-dependent differences in the in vivo hepatic transcriptional response to activation of the AhR by TCDD.
Ovando et al. continue earlier work that evaluated the comparative effects of different dioxins and PCBs, notably TCDD, PeCDF, PCB126, and the non-dioxinlike PCB153 (Vezina et al., 2004). They evaluate the comparative genomic response to acute and subchronic exposure to several dioxinlike compounds (Ovando et al., 2006). This paper expands our understanding of what constitutes a primary response to AhR activation and shows that gene repression in vivo is as much a part of the landscape of activation of the AhR "activation" as the classical models of gene induction. One of the strengths of this study is that these data were generated from animals taken at early time points within the context of two-year cancer bioassays carried out by the National Toxicology Program (National Toxicology Program, 2006; Walker et al., 2005) (http://ntp.niehs.nih.gov). Indeed these animals randomly taken from the two-year cancer study groups at the 13-week interim necropsy. As such, one can directly evaluate these effects in the context that they were at a dose that ultimately was carcinogenic at the end of the bioassay, but that at the time of evaluation was not associated with any major pathology.
One of the problems inherent in many such "descriptive" studies though is deconvoluting what is a primary effect, what is a secondary "adaptive" effect, what is an effect associated with pathology itself, and what is an effect that is a precursor event to the development of a pathology. There is no simple approach to this and no one study can address all aspects of such a question. Ovando et al. focus on whether these responses are a primary effect due to "activation" of the AhR. They show that 61 genes were altered after subchronic exposure to TCDD. Comparative analyses confirm that most of theses changes are induced by dioxins since there was minimal effect of these with exposure to the non-dioxinlike PCB153. Most interestingly was that hierarchical clustering of the data showed that the binary mixture of PCB126 and PCB153 was more like TCDD than PCB126 alone. TCDD, PCB126, and the binary mixture of PCB126/153 are all carcinogenic to the Harlan Sprague Dawley rats and liver as the primary target organ (Walker et al., 2005). They also observed greater effects in the female rats versus male rats that is consistent with the lower potency of TCDD as a tumor promoter in rats as evidenced by differences in hepatocarcinogenicity (Kociba et al., 1978), lower tumor-promoting potential in male rats (Wyde et al., 2002), the inhibitory effect of ovariectomy (Lucier et al., 1991), and the enhancing effect of supplemental estrogens on both tumor promotion and oxidative DNA damage (Wyde et al., 2001a,b). However, at this stage such associations are still not conclusive of a clear linkage.
The authors note that the 13-week time point for this study exhibited minimal pathology, including hepatocyte hypertrophy and multinucleated hepatocytes. However, the observations were nonetheless after subchronic repeated daily exposure. Therefore, the alteration in the observed genes could be due to secondary effects of TCDD rather than a primary effect. Therefore, Ovando et al. followed further after a single acute dose of TCDD, 11 of the 18 transcripts that showed a greater than threefold repression by TCDD after subchronic exposure, to test the hypothesis that these genes were not a consequence of subchronic versus acute exposure. Indeed in all cases, these genes were downregulated after acute exposure to TCDD, albeit it with varying time course. Furthermore, 8 of the 11 genes had identifiable AhR/ARNT-binding sites, further supporting these were direct effects of TCDD. Moreover 8 of these genes exhibited a gender dependence and were repressed in female but not male rats. To evaluate if these genes were regulated directly by the AhR, they analyzed the expression of these genes in AhR knockout mice and showed that seven of these genes required the AhR, again further supporting that the downregulation was a primary response to AhR "activation."
The paper by Boverhof et al. takes on a different challenge, that of understanding the species differences to TCDD and moreover what aspects of the TCDD response are conserved across species, since this would provide insight into likely conserved responses that may occur in humans (Boverhof et al., 2006). To reduce some of the complexity inherent with the interpretation of repeated dose studies, they examined the genomic response after only a single treatment, but with an extensive time course up to 168 h. Through an extensive evaluation of the time course and subsequent dose-response analyses together with dosimetry, histopathology, and clinical pathology, they identify both divergent and conserved responses that in many cases correlate with the effects of TCDD seen in each species. Commonly regulated genes were found associated with chemical/xenobiotic stimulus, nitrogen/amino acid metabolism, and lipid metabolism. Rat-specific genes were identified that were involved in cell growth and lipid metabolism. Conversely, mouse-specific responses were involved in immune function in addition to genes involved in lipid binding/metabolism.
Ultimately, the fundamental application of these findings is to understand the potential biological effects in humans as a consequence of activation of this receptor system, albeit the potential chemotherapeutic application of "modified" AhR ligands, to understanding the human health risk posed by mixtures of persistent AhR ligands. While it is beyond the scope of any two papers to solve the issue of mechanism of action of TCDD, these two papers provide a good stepping stone bridging our extensive understanding of the AhR (Schmidt and Bradfield, 1996) to potential targets that may mediate the toxic effects of TCDD and related compounds.
Armed with this evaluation of the specific targets after acute, subchronic exposure and comparative mouse and rat effects in the liver, now begins the difficult task of synthesizing all these data into a comprehensive model of how these genes and pathways integrate in the manifestation of pathologies and its application to an understanding of the effects of many other ligands, be they the persistent/transient, environmental pollutant, or natural dietary constituents and endogenous metabolites, that have been shown to bind to the AhR. In addition, much of our understanding of the signaling through the AhR has come from the use of the well-characterized, inducible gene CYP1A1. Further examination of the role of the AhR in the regulation of the additional targets identified in these papers may therefore serve to uncover more of the nuances of the AhR-signaling pathway and increase our understanding of the tissue-specific selectivity of action of ligands of the AhR.
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