9.6. MECHANISMS OF DIOXIN ACTION

Knowledge of the mechanisms of dioxin action may facilitate the risk assessment process by imposing bounds on the assumptions and models used to describe possible responses to exposure to dioxin. In this document, the relatively extensive data base on dioxin action has been reviewed, with emphasis on the contribution of the specific cellular receptor for dioxin and related compounds, the Ah receptor, to the mechanism(s) of action. Other reviews referenced in Chapter 2 provide additional background on the subject. Discussion in this chapter will focus on aspects of our understanding of mechanism(s) of dioxin action that are particularly important in understanding and characterizing dioxin risk, including:

n Similarities at the biochemical level between humans and other animals with regard to receptor structure and function;

n Relationship of receptor binding to toxic effects; and

n Role that the purported mechanism(s) of action might contribute to the diversity of biological response seen in animals and, to some extent, in humans.

The remarkable potency of TCDD in eliciting its toxic effects in animals suggested the possible existence of a receptor for dioxin. Biochemical and genetic evidence implicates the TCDD-receptor in the biological responses to dioxin-like compounds. Electrophoretic studies to evaluate the properties of specific proteins from inbred mouse strains reveal the existence of several forms of the TCDD-binding protein. These observations imply the existence of multiple alleles at the Ah locus in mice. The biochemical properties of the different forms of the Ah receptor remain to be described. In particular, the extent to which the different receptor forms affect the sensitivity to TCDD is not known.

Human cells contain an intracellular protein whose properties resemble those of the Ah receptor in animals. Binding studies and hydrodynamic analyses have identified an Ah receptor-like protein(s) in a variety of human tissues. Functional Ah receptors have been found in many human tissues, including lymphocytes, liver, lung, and placenta. By analogy with the existence of multiple receptor forms in mice, it is reasonable to anticipate that the human population will also be polymorphic with respect to Ah receptor structure and function. Therefore, it is also reasonable to expect that humans may differ from one another in their susceptibilities to TCDD. The binding and hydrodynamic properties of the Ah receptor differ relatively little across species and tissues yet responses vary widely; it is impossible, therefore, to account for the diversity of TCDD's biological effects by characteristics of the receptor alone.

TCDD acts via an intracellular protein (the Ah receptor), which is a ligand-dependent transcription factor that functions in partnership with a second protein (known as Arnt); therefore, from a mechanistic standpoint, TCDD's adverse effects appear likely to reflect sustained alterations in gene expression. Mechanistic studies also indicate that several proteins contribute to TCDD's gene regulatory effects and that the response to TCDD probably involves a relatively complex interplay between multiple genetic and environmental factors. Such mechanistic information imposes constraints on the possible models that can plausibly account for TCDD's biological effects and, therefore, on the assumptions used during the risk assessment process. Mechanistic knowledge of dioxin action may also be useful in other ways. For example, knowledge of genetic polymorphisms that influence TCDD responsiveness may allow the identification of individuals at particular risk from exposure to dioxin. In addition, mechanistic knowledge of the biochemical pathways that are altered by TCDD may identify novel targets for the development of drugs that can antagonize dioxin's adverse effects.

As described below, biochemical and genetic analyses of the mechanism by which dioxin induces CYP1A1 gene transcription have revealed the outline of a novel regulatory system whereby a chemical signal can alter the expression of specific mammalian genes. The evidence to date implies that the Ah receptor participates in every biological response to TCDD. For example, studies of structure-activity relationships among congeners of TCDD reveal a correlation between a compound's specific binding affinity and its potency in eliciting biochemical responses, such as enzyme induction. Furthermore, inbred mouse strains in which TCDD binds with lower affinity to the receptor exhibit decreased sensitivity to dioxin's biological effects, such as thymic involution, cleft palate formation, and hepatic porphyria. While there are a few investigators who believe that dioxin may act directly on specific cellular and biological processes without Ah-receptor mediation, the majority of investigators believe that most, if not all, biological responses to dioxin and related compounds are Ah-receptor mediated. A simplified diagram of this hypothesis is presented in Figure 9-2. This hypothesis predicts that TCDD will be found to activate the transcription of other genes via a receptor- and enhancer-dependent mechanism analogous to that described for the cytochrome P4501A1 (CYP1A1) gene.

Compensatory changes, which occur in response to TCDD's primary effects, can complicate the analysis of dioxin action in intact animals. For example, TCDD can produce changes in the levels of steroid hormones, peptide growth factors, and/or their cognate cellular receptors. In turn, such alterations have the potential to produce a series of subsequent biological effects, which are not directly mediated by the Ah receptor. Furthermore, the hormonal status of an animal appears to influence its susceptibility to the hepatocarcinogenic effects of TCDD (Lucier et al., 1991). Likewise, exposure to other chemicals can alter the developmental toxicity of TCDD (Couture et al., 1990). Therefore, in some cases, TCDD may act in combination with other chemicals to produce its biological effects. Such phenomena increase the difficulty of analyzing dioxin action in intact animals

and increase the complexity of risk assessment, given that humans are routinely exposed to a wide variety of chemicals.

The fact that TCDD may induce a cascade of biochemical changes in the intact animal raises the possibility that dioxin might produce a response such as cancer by mechanisms that differ among tissues. These mechanisms are discussed in detail in Chapter 8, along with the supporting biological data and dose-response models. One possible mechanism discussed in Chapter 8 is that TCDD might activate a gene(s) that is directly involved in tissue proliferation. A second mechanism involves TCDD-induced changes in hormone metabolism, which may lead to tissue proliferation secondary to increased secretion of a trophic hormone, and/or to changes in metabolism, which might lead to indirect mutagenic effects. Thus, while this reassessment has identified a number of hypothetical mechanisms for cancer induction by TCDD, there remains considerable uncertainty about which mechanisms occur, with what levels of sensitivity, and in which species. Advances in knowledge regarding the role of such activities in dioxin toxicity will facilitate the development of more definitive biologically based models of dioxin action.

Under some circumstances, TCDD can protect against the carcinogenic effects of polycyclic aromatic hydrocarbons in mouse skin; this may reflect the induction of detoxifying enzymes by dioxin (Cohen et al., 1979; DiGiovanni et al., 1980). In other situations, TCDD-induced changes in hormone metabolism may alter the growth of hormone-dependent tumor cells, producing a potential anticarcinogenic effect (Spink et al., 1990). There is considerable uncertainty about the magnitude and importance of these effects in relation to both dose and response characteristics of dioxins in various species. Nonetheless, these (and perhaps other) effects of TCDD complicate the risk assessment process for dioxin.

A substantial body of biochemical and genetic evidence indicates that the Ah receptor mediates the biological effects of TCDD. This evidence implies that a response to dioxin requires the formation of ligand-receptor complexes. TCDD-receptor binding appears to obey the law of mass action and, therefore, depends on (1) the concentration of ligand in the target cell; (2) the concentration of receptor in the target cell; and (3) the binding affinity of the ligand for the receptor. In principle, some TCDD-receptor complexes will form even at very low levels of dioxin exposure. However, in practice, at some finite concentration of TCDD, the formation of TCDD-receptor complexes may be insufficient to elicit detectable effects. Furthermore, biological events subsequent to TCDD-receptor binding may or may not exhibit a linear response to dioxin. However, recent studies in several laboratories have indicated no evidence of a threshold for relatively simple responses to dioxin-like compounds such as CYP1A1 induction and others. Further information will be required to determine if other responses to dioxin-like compounds requiring gene transcription will also demonstrate low-dose linear behavior.

While much of our understanding of TCDD impacts on genetic activity is derived from studies on liver, studies of other tissues (e.g., skin, thymus) are likely to reveal additional TCDD-responsive genes, which exhibit tissue-specific expression (Sutter et al., 1991). Analyses of the mechanism of dioxin action in such systems appear likely to reveal additional factors that influence the susceptibility of a particular tissue to TCDD. In addition, studies of other TCDD-inducible genes, such as glutathione-S-transferase, quinone reductase, and aldehyde dehydrogenase, may reveal whether differences in enhancer structure, receptor-enhancer interactions, or promoter structure affect the responsiveness of the target gene to TCDD (Whitlock, 1990).

Based on our understanding of dioxin mechanism(s) to date, it is accurate to say that interaction with the Ah receptor is necessary, that humans are likely to be sensitive to the effects of dioxin, and that there is likely to be a variation between and within species and between tissue in individual species based on differential responses to receptor binding. Although threshold mechanisms may exist for some of these responses, thresholds have yet to be demonstrated. Further analyses of dioxin action may provide more insight into the mechanisms by that TCDD and related compounds produce immunological effects, reproductive and/or developmental effects or cancer, effects which are of particular public health concern. A major challenge for the future will be the establishment of experimental systems in which such complex biological phenomena are amenable to study at the molecular level.