TEF concept and Ah receptor

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9.3. SPECIFIC ISSUES

9.3.1. Ah Receptor and Toxicity Factors

Issues relating to the role of the Ah receptor as the common mediator of toxicity of dioxin-like compounds and the cross-species comparability of AhR structure and function frequently arise when the TEF approach is discussed. Recent data relating to each of these issues are discussed below.

9.3.2. The Role of the AhR in the Toxicity of Dioxin-Like Compounds

The general basis for the TEF scheme is the observation that the AhR mediates most if not all of the dioxin-like biological and toxic effects induced by compounds included in the TEF scheme (Safe, 1990; Okey et al., 1994; Birnbaum, 1994; Hankinson, 1995). Binding to the receptor is necessary, but not sufficient, to generate the wide variety of toxic effects caused by dioxin-like halogenated aromatic hydrocarbons (Sewall and Lucier, 1995; De Vito and Birnbaum, 1995) (for additional review references, see Part II, Chapter 2). There are several lines of evidence that the Ah receptor is important in the toxicity of the dioxin-like compounds. A brief discussion of this evidence shall be presented in the following section. Those wishing a more detailed discussion of this issue are referred to Part II, Chapter 2.

Initial studies on the toxicity of PAHs demonstrated that the sensitivity to these compounds varied by strain of mice and segregated with the Ah locus. The Ah locus was then found to encode a receptor designated as the aryl hydrocarbon receptor or AhR. Sensitive strains of mice expressed receptors with high binding affinity for these compounds, while the resistant mice expressed a receptor that poorly bound the PAHs. One of the best ligands for this receptor was TCDD. Shortly after the discovery of the AhR, structure-activity relationship studies demonstrated a concordance between binding affinity to the Ah receptor and toxic potency in vivo in mice. Further support of the role of the AhR in the toxicity of dioxin-like compounds was demonstrated following the development of AhR knockout mice (Fernandez-Salguero et al., 1995; Schmidt et al., 1996; Mimura et al., 1997; Lahvis and Bradfield, 1998). The Ah receptor knockout mice are a strain of mice in which the Ah receptor has been genetically altered so that the receptor is not expressed or "knocked-out" in these mice. Administration of TCDD at doses more than 10 times the LD50 of wild-type mice has not produced any significant dioxin-like effects, either biochemical or toxicological, in the AhR knockout mice (Fernandez-Salguero et al., 1996; Peters et al., 1999). These data as a whole demonstrate that the binding to the AhR is the initial step in the toxicity of dioxin-like compounds.

9.3.3. Species Comparison of the AhR

Although binding to the AhR initiates a cascade of molecular and cellular events leading to toxicity, the exact mechanism of action of dioxin-like compounds is not completely understood. One difficulty in determining the mechanism is our limited understanding of the normal physiological role of the AhR, which would aid in understanding of potential species differences in response to dioxin-like chemicals. The available data indicate that the AhR does play an important role in normal processes and that there are a number of similarities in the action of the AhR between species. These data strengthen our confidence in species extrapolations with these chemicals.

There are several lines of evidence suggesting that the AhR is an important factor in developmental and homoeostatic processes. The AhR is a ligand-activated transcription factor that is a member of the basic-helix-loop-helix-Per-Arnt-Sim (bHLH-PAS) superfamily. The bHLH-PAS superfamily consists of a growing list of at least 32 proteins found in diverse organisms such as Drosophila, C. elegans, and humans. Many of these proteins are transcription factors that require either hetero- or homodimerization for functionality. These proteins regulate circadian rhythms (per and clock) and steroid receptor signaling (SRC-1, TIF2, RAC3) and are involved in sensing oxygen tension (Hif-1, EPAS-1/HLF) (Hahn, 1998). The AhR is also a highly conserved protein that is present in all vertebrate classes examined, including modern representatives of early vertebrates such as cartilaginous and jawless fish (Hahn, 1998). In addition, an AhR homologue has been identified in C. elegans (Powell-Coffman et al., 1998). The classification of the AhR as part of the bHLH-PAS superfamily and its evolutionary conservation imply that this protein may play an important role in normal physiological function. It has been proposed that understanding the function of the bHLH-PAS family of proteins and the phylogenetic evolution of the AhR may lead to an understanding of the role of this protein in normal processes (Hahn, 1998).

The process of development is a complex phenomenon that involves the specific expression of numerous genes in a spatial and temporal pattern. The importance of a particular gene in developmental biology is often inferred by its spatial and temporal expression during development. The AhR is expressed in a tissue, cell, and temporal pattern during development (Abbott et al., 1995). It is highly expressed in the neural epithelium, which forms the neural crest (Abbott et al., 1995). The expression of the AhR at critical periods during development suggests that this protein has important physiological functions.

Further evidence of the role of the AhR in developmental processes is provided by the development and study of AhR knockout mice. Three strains of AhR knockout mice have been produced using a targeted disruption of the AhR locus (Fernandez-Salguero et al., 1995; Schmidt et al., 1996; Mimura et al., 1998; Lahvis and Bradfield, 1998). The AhR -/- mice develop numerous lesions with age (Fernandez-Salguero et al., 1995). Mortality begins to increase at about 20 weeks, and by 13 months almost half of the mice either die or become moribund. Cardiovascular alterations consisting of cardiomyopathy with hypertrophy and focal fibrosis, hepatic vascular hypertrophy and mild fibrosis, gastric hyperplasia, T-cell deficiency in the spleen, and dermal lesions are apparent in these mice and the incidence and severity increases with age (Fernandez-Salguero et al., 1995). Although male and female AhR -/- mice are fertile, the females have difficulty maintaining conceptus during pregnancy, surviving pregnancy and lactation, and rearing pups to weaning (Abbott et al., 1999). It should be noted that the AhR knockout mice are resistant to the toxic effects of TCDD.

Comparisons between the AhR of experimental animals (primarily rodents) and the human AhR have revealed a number of similarities in terms of ligand and DNA binding characteristics and biochemical functions. Tissue-specific patterns of expression of AhR mRNA are similar in rats, mice, and humans, with highest levels generally detected in lung, liver, placenta, and thymus (Dolwick et al., 1993; Döhr et al., 1996). Nuclear AhR complexes isolated from human and mouse hepatoma cells (Hep G2 and Hepa 1c1c7, respectively) have similar molecular weights. Although the human AhR appears more resistant to proteolytic digestion by trypsin or chymotrypsin, the major breakdown products were similar between the two species, and photolabeling analysis with TCDD suggested common features in the ligand binding portion of the receptors (Wang et al., 1992).

Limited analysis has suggested the average human AhR exhibits a lower binding affinity for various HAHs than "responsive" rodent strains. However, similar to a variety of experimental animals, human populations demonstrate a wide variability in AhR binding affinity (Micka et al., 1997). Recent determination of AhR binding affinity (Kd) toward TCDD in 86 human placenta samples showed a greater than twenty-fold range in the binding affinity, and this range encompasses binding affinities similar to those observed in sensitive and resistant mice (Okey et al., 1997). Whereas the concentration of various ligands required to activate a human AhR reporter gene construct was higher than required with rodent cell cultures, the actual rank order of binding affinities was in agreement (Rowlands and Gustafsson, 1995). Although comparisons have been made of the TCDD binding affinity to the AhR of different species, caution should be used when attempting to predict species sensitivity to TCDD and related compounds. For mice, the sensitivity to the biochemical and toxicological effects of TCDD and related compounds is associated with the relative binding affinity of TCDD to the AhR in the different strains (Birnbaum et al., 1990; Poland and Glover, 1990). However, the relative binding affinity of TCDD to the AhR across species does not aid in the understanding of interspecies differences in the response or sensitivity to TCDD (DeVito and Birnbaum, 1995).

The human AhR also demonstrates other slight differences when compared to the AhR from experimental animal species. The molecular mass of the human AhR ligand-binding subunit appears to be greater than the AhR subunit from certain TCDD "responsive" mouse strains but similar to the receptor molecular mass for rats (Poland and Glover, 1987). Currently there has been no association established between differences in the molecular mass of the AhR and sensitivity to a particular biochemical or toxicological response across species (Okey et al., 1994). The non-liganded human AhR appears thermally more stable compared to AhR from various rodent species, whereas the reverse situation exists with the liganded human AhR (Nakai and Bunce, 1995). Transformation of the ligand-bound human AhR receptor (isolated from colon adenocarcinoma cells) to the DNA-binding state, unlike rodent hepatic AhR, is temperature dependent (Harper et al., 1992). The importance of these species differences in transformation and stability of the AhR in the species sensitivity to TCDD remain uncertain. However, in critical areas of receptor function such as ligand recognition, transformation, and interaction with genomic response elements, the human AhR is comparable to the AhR isolated from experimental animals.

Ligand-bound or transformed AhR from a variety of mammalian species, including humans, bind to a specific DNA sequence or "dioxin response element" with similar affinities (Bank et al., 1992; Swanson and Bradfield, 1993). The bHLH structure of receptor proteins such as AhR ensures appropriate contact and binding with DNA recognition sites. Amino acid sequence analysis between mouse and human AhR shows an overall sequence homology of 72.5%, whereas the bHLH domain shows 100% amino acid concordance (Fujii-Kuriyama et al., 1995). In comparison, the deduced amino acid composition of the AhR from killifish was 78%-80%, similar to the amino acid sequence of rodent and human AhR (Hahn and Karchner, 1995). These studies demonstrate a concordance between the structure of the receptor and its function across species.

The majority of scientific evidence to date supports the theory that binding to the AhR is a necessary first step prior to dioxin-like compounds eliciting a response, as discussed in Part II, Chapter 2. Current research has identified the AhR in a variety of human tissues and cells that appear to function in a similar manner to the AhR from experimental animals, including fish, birds, and mammals. When multiple endpoints are compared across several species, there exists a high degree of homogeneity in response and sensitivity to TCDD and related compounds (DeVito et al., 1995). Therefore, these data provide adequate support for the development of the TEF methodology. However, these data also reflect the true complexity of intra- and interspecies

comparisons of biochemical and toxicological properties. Continued research into the variety of additional cytoplasmic and nuclear proteins capable of interacting with the AhR signaling pathway will ultimately lead to a better understanding of the observed species and strain variability in the response to dioxin-like chemicals and may be useful in further refining TEFs.

9.3.4. Mode of Action and Implications for the TEF Methodology

Many of the toxic effects of dioxins are mediated by disruption of normal growth and differentiation processes. For example, TCDD alone is capable of producing cancer in experimental animals. However, its genotoxicity is limited. From an operational point of view, TCDD is a tumor promoter (See Part II, Chapter 6). Tumor promoters act by disrupting the natural balance between cell replication and cell death. Similarly, many of the non-cancer effects, such as immunotoxicity and developmental toxicities, are due to TCDD-induced alterations in cell growth and differentiation. While these events are initiated by the activation of the Ah receptor, the exact molecular and cellular alterations beyond receptor binding remain uncertain. One criticism of the TEF methodology is that the exact molecular mechanisms for the toxic effects of these chemicals is uncertain and thus one cannot apply this method to mixtures with certainty. The uncertainties in understanding the exact molecular mechanism of dioxin action is not unique and does not detract significantly from the utility of the TEF methodology. The exact molecular mechanisms of the biochemical and physiological effects of estrogens are also uncertain. This does not decrease our confidence that if a chemical binds to the estrogen receptor and induces uterine growth in vivo that the chemical is estrogenic and that it can be useful to describe its potency relative to estradiol. Similarly, if a chemical binds to the Ah receptor and induces dioxin-like effects, we can classify the chemical as dioxin-like and describe its relative potency to TCDD without understanding every molecular event leading to the biological effect. For many of the chemicals assigned TEF values, there are in vitro Ah receptor binding data and a number of in vivo studies estimating the REP of these chemicals for toxic and biochemical effects.

See also

• TEF concept
• Ah receptor ligands
• TEF concept in environmental health assessment
• TEF concept and uncertainty analysis
• Additivity of TEFs
• TEF concept and ecology
• TEF concept and Ah receptor
• Estimating TEF values