Joyce C. Knutson

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.

Electroporation: Parameters affecting transfer of DNA into mammalian cells☆

Electroporation, the reversible breakdown of cell membranes caused by a high-voltage discharge, is a rapid, simple, and efficient method for introducing DNA into mammalian cells. An instrument for electroporation which permits the high-voltage discharge waveform to be varied with respect to rise time, peak voltage, and fall time is described. The uptake and expression of SV40 DNA following electroporation of two cell types, a human carcinoma-derived cell line, HEp-2, and a human lymphoblastoid cell line, 721, depended on the peak voltage and the fall time of the voltage discharge. The electronic parameters which produced optimum DNA transfer, however, differed for the two cell types. DNA as large as 150 kb was introduced into cells by electroporation. Cells can be electroporated in either phosphate-buffered saline or culture medium containing fetal bovine serum, and the efficiency of DNA transfer does not vary with cell densities from 10(6) to 2 X 10(7)/0.5 ml. Exposing the cells to multiple voltage discharges did not improve DNA transfer. DNA has been introduced by electroporation into all cell types tested, including human carcinoma-derived cell lines, human lymphoblastoid cell lines, human fibroblast strains, and primary human lymphocytes. To obtain maximal DNA transfer by this method, however, one must optimize the peak voltage and fall time of the discharge waveform for each cell type.

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.

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.

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.