Alan Poland

Response of murine epidermis to 2,3,7,8-tetrachlorodibenzo-p-dioxin: Interaction of the Ah and hr loci

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) and related halogenated aromatic hydrocarbons produce epidermal hyperplasia, hyperkeratosis and sebaceous gland metaplasia in the skin of mice bearing the recessive mutation (hr/hr) hairless. This response is mediated through the cytosol receptor protein: the structure-activity relationship for receptor binding corresponds to that for production of the skin lesion, and these histopathological changes segregate with the genetic polymorphism at the Ah locus, the locus determining the cytosol receptor. In HRS/J mice, an inbred strain segregating for the hr locus, both hairless (hr/hr) and haired (hr/+) mice possess the high-affinity cytosol receptor and respond to TCDD with the induction of epidermal aryl hydrocarbon hydroxylase activity, a receptor-mediated biochemical response; however, only hr/hr mice develop the proliferative/metaplastic skin response. We propose a genetic model for the interaction of the Ah and hr loci, to account for the differential response to TCDD observed in the skin of HRS/J hr/hr and hr/+ mice.

Keratinization of mouse teratoma cell line XB produced by 2,3,7,8-tetrachlorodibenzo-p-dioxin: An in vitro model of toxicity

Abstract 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD), an extremely potent toxin, serves as the prototype of a large group of halogenated aromatic hydrocarbons, all of which produce a similar and characteristic pattern of toxic lesions, which includes hyperkeratosis and squamous metaplasia in the skin; induce a battery of coordinately expressed enzymes, the most studied of these being aryl hydrocarbon hydroxylase (AHH) activity; and appear to exert both their toxic effects and enzyme induction through stereospecific, reversible binding to a cytosolic receptor protein. Previous studies performed with over 30 cell types have failed to demonstrate any in vitro toxicity from TCDD. When XB cells derived from a mouse teratoma are cultured at high density (to prevent spontaneous differentiation) along with lethally irradiated 3T3 cells, the addition of TCDD produces a dose-related keratinization response as detected by red staining with Rhodanile blue. Maximal keratinization is produced by 5 × 10 −11 M TCDD. This is a direct effect of TCDD on XB cells, and will occur in the absence of the feeder cells if the teratoma cells are cultured in 3T3-conditioned media. The keratinization produced by TCDD was examined by electron microscopy and histologic staining, and appears to be similar to that which occurs spontaneously when XB cells are plated at low density. XB cells contain the cytosol receptor and respond to TCDD with a dose-related induction of AHH activity. The potencies of halogenated aromatic hydrocarbon congeners [dibenzo-p-dioxins, dibenzofurans, biphenyls and azo(xy)benzenes] to produce keratinization in the XB/3T3 system corresponds to their binding affinities for the cytosol receptor. Nonhalogenated aromatic hydrocarbons, which are agonists for the receptor [such as benz(a)anthracene and 5,6-benzoflavone], also produce a dose-related keratinization in XB/3T3 cultures, whereas unrelated toxins (such as alkylating agents and inhibitors of nucleic acid synthesis) do not. Our results suggest that the keratinization produced by TCDD and congeners in XB/3T3 cultures is mediated by the cytosolic receptor, and that this system provides an in vitro model for the in vivo toxic effects produced by halogenated aromatic hydrocarbons in the epidermis.

Variation in the molecular mass of the Ah receptor among vertebrate species and strains of rats

The Ah receptor in eight vertebrate species was characterized by labeling the cytosolic fraction of tissue with the photoaffinity ligand, [125I]-2-azido-3-iodo-7,8-dibromodibenzo-p-dioxin, and analysis of the products by denaturing gel electrophoresis. The apparent molecular mass of the dominant labeled peptide showed appreciable species variation: mouse-95 kDa; chicken (embryo)-101 kDa; guinea pig-103 kDa; rabbit-104 kDa; rat-106 kDa; human-106 kDa; monkey-113 kDa, and hamster-124 kDa. Seven inbred strains of rats, had a Ah receptor ligand binding peptide of 106 kDa; however outbred Long-Evans rats were shown to be polymorphic expressing a 101 kDa and/or 106 kDa allelic forms. The notable frequency of structural variation in the Ah receptor is in contrast to the analogous highly conserved steroid hormone receptors.

2,3,7,8-Tetrachlorodibenzo-p-dioxin: Failure to demonstrate toxicity in twenty-three cultured cell types☆

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD), one of the most potent small molecule toxins known was investigated for its toxic effect on 23 cultured cell types, including primary cultures and cells from established and transformed cell lines. The cells, derived from tissues and/or species susceptible to TCDD toxicity in vivo, were exposed to TCDD for an extended period, usually a week or longer. Examination of the cells for altered morphology, decrease in percentage viability, or diminished growth rate revealed no toxic effect of TCDD in any cell type, including those which respond to TCDD with the induction of aryl hydrocarbon hydroxylase activity.

The Ah receptor in marine animals: phylogenetic distribution and relationship to cytochrome P4501A inducibility

Abstract In mammals, the induction of cytochrome P4501A forms by chlorinated dibenzo-p-dioxins, chlorinated dibenzofurans, and halogenated biphenyls is under control of a soluble protein known as the Ah (aromatic hydrocarbon) receptor. Little is known about the presence and properties of the Ah receptor in other vertebrate and invertebrate species. In these studies, we sought evidence for an Ah receptor in the liver or liver-equivalent of 20 species of marine and freshwater animals, using the photoaffinity ligand 2-azido-3-[ 125 I] iodo-7,8-dibromodibenzo-p-dioxin (N 3 [ 125 I]Br 2 DD). Specific labeling of cytosolic proteins by N 3 [ 125 I]Br 2 DD was observed in seven species of teleost and elasmobranch fish, in PLHC-I fish hepatoma cells, and in beluga whales. No specifically labeled proteins were found in cytosol from two species of agnathan fish nor in any of nine invertebrate species representing eight classes of four phyla. The presence or absence of specifically labeled polypeptides corresponds with the inducibility of cytochrome P4501A and sensitivity to the toxic effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin and related planar halogenated aromatic hydrocarbons in many of these groups. Thus, Ah receptor function may have arisen early invertebrate evolution and has been conserved from elasmobranch and teleost fish to mammals.

Ca2+-dependent proteolysis of the Ah receptor.

We previously reported (J. Biol. Chem. (1986) 261, 6352-6465) that the photoaffinity ligand for the Ah receptor, [125I]-2-azido-3-iodo-7,8-dibromodibenzo-p-dioxin, upon incubation with the liver cytosol fraction from C57BL/6 mice, labeled in a 1:1 ratio two peptides that had apparent molecular masses of 95 and 70 kDa and similar proteolytic fragmentation patterns. In the cytosolic fraction of Hepa 1 cells, a cloned murine hepatoma cell line, the product of photoaffinity labeling is almost exclusively a 95-kDa peptide which is rapidly hydrolyzed by a Ca2+-dependent proteinase to a 70-kDa peptide as well as other fragments. Thus, the ligand binding unit of the Ah receptor in C57BL/6 mouse liver and Hepa 1 cell is a 95-kDa peptide, and the 70-kDa fragment is a proteolytic artifact. The Ca2+-dependent proteinase which hydrolyzes the 95-kDa peptide has the properties of calpain II: (i) an absolute requirement for Ca2+, with maximal activity at 0.5 to 1.0 mM Ca2+; (ii) a pH optimum of 7.5 to 8.0; (iii) inhibition by EDTA, iodoacetamide, leupeptin and L-trans-epoxysuccinylleucylamido(4-guanidino)butane, but not by soybean trypsin inhibitor, aprotinin, or phenylmethanesufonyl fluoride. Upon chromatographic separation of the liver cytosol of C57BL/6 mice on DEAE-Sephacel, Ca2+-dependent proteinase activity (using casein or the labeled 95-kDa peptide as substrates) elutes with 0.25 M NaCl, and a specific proteinase inhibitor elutes with 0.15 M NaCl. Ca2+-dependent proteinase activity that hydrolyzes the 95-kDa peptide is found in the liver cytosols of several mammalian species.

The trouble with TEFs.

Comments on Van den Berg, et al. Toxic equivalency factors (TEFs) for PCBs, PCDDs, PCDFs for humans and wildlife. Environ Health Perspect 106:775-792 (1998)

A Consideration of the Mechanism of Action of 2,3,7,8-Tetrachloro-Dibenzo-P-Dioxin and Related Halogenated Aromatic Hydrocarbons

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is the prototype of a group of halogenated aromatic hydrocarbons which produce a similar and characteristic pattern of toxic and biochemical responses. The most well-studied biochemical response to these compounds is the induction of activity of the microsomal monooxygenase, aryl hybro-carbon hydroxylase (AHH). The induction of AHH activity by TCDD and congeners is mediated through their stereospecific, reversible binding to a cytosol protein, the induction receptor. Two types of evidence indicate that the toxic responses produced by these compounds are also mediated through the cytosol receptor: a) the structure-activity relationship for halogenated aromatic hydrocarbon congeners to bind to the receptor corresponds to that for their toxic potency: and b) several toxic responses produced by TCDD in mice, segregated with the Ah locus, the genetic locus which determines the receptor. While toxicity is mediated through the receptor, many tissues in various animal species and numerous cell types in vitro, contain the receptor and respond with the induction of AHH activity when challenged with TCDD, but do not display toxic responses.

2,3,7,8-Tetrachlorodibenzo-p-dioxin: Toxicity in vivo and in vitro

ABSTRACT 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is the prototype of a group of halogenated aromatic hydrocarbons, a class of potent toxic and teratogenic compounds. All the compounds 1) are approximate isosteroeomers, 2) produce a characteristic pattern of toxic responses, 3) induce a common set of enzymes, including aryl hydrocarbon hydroxylase (AHH) activity, and 4) compete for a high affinity binding site of a cytosol protein, which is the receptor for enzyme induction. The rank order of binding affinities of the halogenated aromatic hydrocarbons for the receptor correspond very well with their toxic potencies as well as their potencies as inducers of AHH activity. The receptor is determined by the Ah locus. C57BL/6J mice which have the high affinity receptor are ten times more sensitive to AHH induction by TCDD than DBA/2J mice which have a lower affinity receptor. Two toxic responses to TCDD, thymic atrophy and cleft palate formation, segregate with the Ah locus. This genetic segregation of toxicity, and the correspondence of the structure-activity relationship (SAR) for receptor binding and for toxic potency indicate that toxicity is mediated by the receptor. Although binding to the receptor is necessary for toxicity, it is not sufficient, for no toxic effect of TCDD was observed in twenty-three cultured cell types, including many which have the receptor. TCDD does produce a dose-related keratinization of a cell line derived from a teratoma, and the SAR for the keratinization response corresponds with that for toxic potency. A single cell type in vitro , therefore, is sufficient to produce a toxic response to TCDD.

Photoaffinity labelling of the Ah receptor.

A series of halodibenzo-p-dioxins bearing the arylazide photolabile functional group were synthesized and tested as photoaffinity labels for the Ah receptor. 2-Azido-3-iodo-7,8-dibromodibenzo-p-dioxin (KD = 0.76 X 10(-9) M) was selected for radiosynthesis. Analysis of the 125I-photoaffinity-labelled proteins in mouse-liver cytosol by denaturing gel electrophoresis revealed two peptides which had apparent molecular masses of 95,000 and 70,000 daltons respectively, were labelled in an approximately 1:1 ratio and were selectively labelled at low concentrations of the photoaffinity ligand (0.05 KD = 0.04 X 10(-9) M). In addition, their labelling was inhibited by co-incubation with an excess of unlabelled ligand. On chromatographic separation under non-denaturing conditions, these two peptides co-migrated. These studies suggest that the Ah receptor in mouse liver cytosol is a heterodimer composed of two non-covalently bound peptides (95 K and 70 K) which each have a ligand binding site.