Biochemistry And Toxicity 151 Biological Persistence

As noted earlier, the central event that instigates the biological effects of TCDD and dioxinlike substances is thought to be their binding to a receptor protein. This aryl hydrocarbon (Ah) receptor (AhR; see Chapter 12) was first postulated during studies of PCDDs and polycyclic aromatic hydrocarbons (PAHs) such as 3-methylcholanthrene.89-9i Sequencing of the gene for the AhR revealed a surprise. Rather than being a member of the steroid hormone receptor family as supposed originally, it belongs to a family of basic helix-loop-helix proteins containing a sequence known as the PAS domain.92

Once a compound is bound to the AhR, a series of intracellular processes may ensue, including shedding and binding of other factors [including the aryl hydrocarbon nuclear translocator (ARNT)], migration into the nucleus, and binding of the complex to specific sequences of DNA called AhR-responsive elements. By influencing the rate of transcription of specific messenger RNAs, the rate of synthesis of the related proteins is altered. Thus, the binding of an appropriate compound (ligand) to the receptor can change the cellular concentration of certain proteins by regulating the expression of genes governing their synthesis.93 The presence or absence of cofactors may account for differences in gene expressions by tissue. Other molecular mechanisms are of increasing interest.92-94

Among the proteins induced by the Ah receptor are three cytochrome P450 enzymes: CYPIA1, CYPIA2, and CYPIB1. These phase I enzymes oxidize "foreign" (xenobiotic) substances, including PAHs, plant constituents such as flavones, aromatic amines, and some pharmaceutical drugs. One consequence of this metabolic conversion is that the xenobiotic substance, typically lipid- rather than water-soluble, is then subject to further enzymatic conversion. Phase II enzymes add hydrophilic groups, enhancing water solubility and excretion from the body. In this manner, AhR-induced enzymes can reduce the biological effect of some environmental agents by facilitating their metabolic degradation. 95-96

On the other hand, the oxidative transformation of a xenobiotic compound by the AhR-induced cytochrome P450 enzymes may greatly enhance its biological activity.95 A classical example is 2-acetylaminoflourene (AAF), a potent liver carcinogen in rats. A number of studies have shown that the proximal carcinogen is not AAF but an oxidized metabolite produced via CYPIA2.95,97 Preparations containing these enzymes are used in microbial mutagenesis tests to activate otherwise inert genotoxins.

When the oxidative degradation of the xenobiotic compound facilitates its excretion so that the intracellular concentration is reduced, a negative feedback is established: Binding of the compound to the receptor is also reduced, transcription decreases, the level of the cytochrome P450 enzymes diminishes, and the system returns to its initial condition. Other sources of feedback may also be present.92

TCDD and related halogenated compounds strongly induce CYPIA1 and CYPIA2 but are not readily oxidized by these enzymes. They are apparently protected from attack by the presence of halogen atoms in certain positions of the molecule. Hence, they are excreted very slowly, resulting in a prolonged and amplified response. In effect, a feedback system that governs behavior of other AhR-binding substances (such as the PAHs) is inoperative in the case of TCDD. Thus, the extraordinary biological potency of dioxinlike substances may be due to the consequence of their unique combination of two properties:

a high affinity for the Ah receptor and biological persistence. The relevance of persistence is evident from a comparison of the behavior of dioxinlike compounds and PAHs.* Although some PAHs bind to the Ah receptor with an affinity almost equal to that of TCDD, their in vivo potency (as measured by enzyme induction) is many orders of magnitude less.98

1.5.2 Perturbation of Hormones and Growth Factors

Although the induction of CYPIA1 is the best characterized of the biochemical effects of dioxinlike compounds, it is by no means the only one. The expression of a growing number of genes are thought to be regulated by the Ah recep-tor.99,100 This may provide one mechanism whereby dioxinlike compounds perturb the regulation of hormones, growth factors, and other molecular messengers that control growth and differentiation with diverse and potentially devastating impact. Some examples follow.

TCDD may alter the levels of certain hormones through its influence on the enzymes that metabolize primarily xenobiotic compounds. For instance, TCDD induces one form of UDP-glucuronosyltransferase (UDPGT), a phase II enzyme that increases a chemical's solubility by adding glucuronic acid. In addition to xenobiotics, UDPGT also conjugates and enhances the excretion of thyroxine (T4), causing reduced serum levels of this thyroid hormone in rats. 101 Among the resultant complications is a perturbation of an important biological feedback system: The pituitary responds to low T4 with increased secretion of thyroid-stimulating hormone (TSH). When prolonged, this may lead to thyroid tumors, 102 a sensitive endpoint in TCDD-exposed rats.103

TCDD does not bind to steroid hormone receptors, and steroid hormones do not bind to the Ah receptor. 104 Nevertheless, TCDD affects steroid hormone regulation in more subtle ways. Thus, TCDD decreases (downregulates) the number of estrogen receptors in certain organs of the female rodent, making tissues less responsive to this hormone. 105 This may decrease both fertility and incidence of tumors of these organs, as has been suggested in rats exposed to TCDD postnatally.106

TCDD reduces testosterone levels in adult male rats by decreasing the production of testosterone from cholesterol in the testes at a critical rate-limiting step. The pituitary (and/or hypothalamus) normally responds to low testosterone concentration by increasing secretion of luteinizing hormone, causing increased production of testosterone. TCDD interferes with this feedback system, preventing the compensatory increase of luteinizing hormone. 107-110

TCDD also affects growth factors, a class of extracellular signaling molecules. In the female rat liver, TCDD may increase migration of epidermal

* Recent work shows that changing a single nucleotide flanking the AhR-responsive element can determine whether a gene is transcribed by AhR bound to PAH or TCDD. This finding may have important implications for both understanding molecular mechanisms and extension of TEFs to other classes of compounds [see T. Matikainen et al., Nature Genetics 28: 355-360 (2001)].

growth factor receptors (EGFRs) internally from the cell membrane, providing a stimulus for mitosis.i°3.i04,iii This effect appears to depend on ovarian hormones; interactions between EGFRs and estrogen receptors have been noticed elsewhere.u2 TCDD may affect EGFRs by increasing the levels of transforming growth factor a (TGFa), a ligand for EGFRs. i°3 In mice, TCDD alters the differentiation of certain tissues in the developing palate. This may be caused by perturbation of growth factors and their receptors, including EGFRs. The palatal shelves come into contact but fail to fuse, resulting in cleft palate. U3

TCDD can lead to increased phosphorylation of amino acids. Protein kin-ases often play important roles in transducing signals across cell membranes, regulation of growth factor receptors, and cell differentiation. "4 TCDD may alter regulation of the cell cycle.^-ii5 TCDD also influences a number of other chemical messengers, including the glucocorticoid hormone receptor, plasminogen activator inhibitor, protein kinase C, interleukin-1b, and other cytokines."

TCDD has been called a persistent environmental hormone.n® One of its molecular mechanisms—binding to a receptor that regulates gene expression— has certain similarities to the action of steroid hormones^ as well as important differences.92 It alters cell growth and differentiation. It affects other hormones and growth factors, including altering the levels of their receptors. Finally, like hormones, TCDD causes significant effects at very low doses. This knowledge of dioxin's biochemistry increases our concern over its widespread occurrence in the environment.

Does the body possess some unidentified hormone that binds to the Ah receptor, serving an important but unknown function? Such a situation is not unprecedented. A number of such orphan receptors (i.e., receptors without known ligands) have been found.U8 Dioxin may be a case of toxic mimicry, possessing a molecular shape similar to that of its putative natural counterpart. The long residence time of TCDD in the body may alter expression of AhRregulated genes for an inappropriately long period of time. It is also possible that the supposed natural ligand of the Ah receptor might normally function during a specific period of development; TCDD may activate the system at the wrong time.u9 Knockout mice—animals without a functional AhR—are viable but show defects in the development of the liver, immune system, and repro-duction.i2°-i22 The question of the normal function of the AhR is a major goal of molecular research in the field.

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