Biomedical subjects

T A Gasiewicz

Publications and source records attributed to T A Gasiewicz.

At least 55 records · Page 3Linked to original sources

PubMed1994

Dexamethasone, beta-estradiol, and 2,3,7,8-tetrachlorodibenzo-p-dioxin elicit thymic atrophy through different cellular targets.

The effects of single doses of dexamethasone (DEX), beta-estradiol-17-valerate (E2), and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on the kinetics of thymic atrophy and related bone marrow and thymocyte phenotype alterations were examined. The results imply differences in the mechanisms by which these compounds act. Of the three compounds, DEX induced maximal atrophy by 3 days with complete recovery by Day 12. At the point of maximal atrophy, the RAG-1+TdT+CD4+8+3int thymocyte population was proportionately the most depleted. In contrast, TCDD and E2 caused maximal thymic atrophy by Day 12. E2 treatment, like DEX, resulted in a preferential decrease in the RAG-1+TdT+CD4+8+3int population, but unlike DEX, this decrease persisted. TCDD-induced thymic atrophy resulted from a proportional loss of all classes of thymocytes. There was no significant relative reduction of TdT+RAG-1+ cells by TCDD in the thymus. A slow and persistent reduction of TdT and RAG-1 in bone marrow by both TCDD and E2 contrasted with the rapid reduction and quick recovery of these markers in marrow from DEX-treated animals. Additional studies showed that only DEX-induced atrophy was accompanied by the induction of thymocyte apoptosis, as detected by multiple nucleosomal length DNA fragments within the first 24 hr. The different kinetics and proportions of subsets in the atrophied thymuses, as well as the distinct patterns of alterations of RAG and TdT expression, and the presence or the absence of apoptosis provide evidence for different mechanisms of thymic atrophy by these agents. The slow induction and longer persistence of thymic atrophy induced by E2 and TCDD, as well as their effects on bone marrow stem cell markers, suggest that bone marrow thymocyte precursors are major targets for these agents.

Animals↗

PubMed1994

Alternate immune system targets for TCDD: lymphocyte stem cells and extrathymic T-cell development.

We here summarize evidence that thymic atrophy induced by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) can be mediated, at least in part, by damage to extrathymic T-cell precursors in bone marrow and fetal liver. This atrophy induction does not involve apoptotic mechanisms in thymocytes affected by the bcl-2 proto-oncogene. TCDD mediates atrophy induction through its specific receptor (the AhR) and not through effects on the estrogen receptor. Both TCDD and estradiol induce extrathymic T-cell differentiation in the liver. These extrathymic T-cell populations include cells expressing elevated levels of V beta T-cell receptors that are normally deleted in thymic development.

Animals↗

PubMed1994

Purification to homogeneity of the heteromeric DNA-binding form of the aryl hydrocarbon receptor from rat liver.

The aryl hydrocarbon receptor (AhR) is a transcriptional enhancer that is activated by the binding of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and related toxic xenobiotics, as well as some naturally occurring compounds. Ligand binding initiates 1) dissociation of the ligand-bound monomeric AhR from the ligand-unoccupied multimeric complex and 2) biochemical and/or conformational changes that enable association of the ligand-bound monomer with other proteins. This heteromeric complex has high affinity for specific elements [dioxin-responsive elements (DREs)] in the regulatory regions of a number of structural genes, the induction and/or repression of which may be a mechanism of toxicity of TCDD. We have developed a relatively simple and rapid procedure that enables purification to homogeneity of a TCDD-bound receptor complex. The final step of purification is based on binding to an oligonucleotide containing the specific DRE sequence that is found in the upstream region of the CYP1A1 structural gene. The purified complex retains in vitro DRE-binding function. Silver staining and Western blot analyses demonstrate that the complex consists of the AhR ligand-binding monomer of approximately 104 kDa, plus two proteins (94 and 96 kDa) that are recognized by antibodies prepared against the AhR nuclear translocator protein. Previous attempts to purify a DRE-binding form of the AhR were unsuccessful because of dissociation of the complex during chromatography; this is the first report of an isolated functional complex. The purified preparation will be valuable in further studies of receptor regulation and function.

Animals↗

PubMed1993

Transformation of the aryl hydrocarbon receptor to a DNA-binding form is accompanied by release of the 90 kDa heat-shock protein and increased affinity for 2,3,7,8-tetrachlorodibenzo-p-dioxin.

The binding of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) to the aryl hydrocarbon receptor (AhR) elicits a sequence of poorly defined molecular events that ultimately yield a heteromeric transformed AhR that is active as a transcription factor. We have previously developed a model of the ligand-initiated transformation of the AhR to the DNA-binding state based on characterization of several forms of the AhR with respect to their physicochemical properties and DNA-binding affinities. The present studies were designed to determine whether, and at what stage, this process of transformation alters the receptor's affinity for TCDD. In rat hepatic cytosol, approx. 10% of the TCDD specifically bound to the AhR rapidly dissociated (t1/2 approximately 1 h), while the remainder was only slowly dissociable (t1/2 approximately 70 h). The isolated DNA-binding forms of the receptor (monomeric and transformed) bound TCDD very tightly (t1/2 > 100 h), whereas TCDD was dissociable from the non-DNA-binding receptor form(s). A lower incubation temperature (0-4 degrees C) and the presence of molybdate partially stabilized the non-DNA-binding fraction of the TCDD.receptor complex and also enhanced TCDD dissociation in crude cytosol. Immunoprecipitation of the different AhR forms with an anti-AhR antibody and immunoblotting with antibody to the 90 kDa heat-shock protein (hsp90) demonstrated that hsp90 was associated with the unoccupied receptor complex as well as with a fraction of the non-DNA-binding TCDD.receptor complex; isolated DNA-binding forms did not contain detectable hsp90. We conclude that while hsp90 remains associated with the AhR, TCDD is readily dissociable; following release of hsp90, however, TCDD becomes very tightly bound, and remains so upon completion of transformation.

Animals↗

PubMed1993

The human estrogen receptor structural gene contains a DNA sequence that binds activated mouse and human Ah receptors: a possible mechanism of estrogen receptor regulation by 2,3,7,8-tetrachlorodibenzo-p-dioxin.

TCDD decreases ER levels in vitro and in vivo, possibly through down-regulation of the ER structural gene, although the actual mechanism is unknown. We report the identification of six partial and two full length DREs in the 5' flanking and coding regions of the human ER gene. To test the ability of the coding region DRE (+102) to bind the activated AhR, two complementary oligonucleotides corresponding to the DRE-containing region of the human ER gene were synthesized, termed hERO, and used in a gel shift assay. TCDD-activated nuclear extracts from both mouse Hepa 1c1c7 cells and human MCF-7 cells specifically bound to the hERO with relative binding affinities of 15.5 nM and 5.6 nM, respectively. This binding was dependent upon a functional DRE. The hERO also specifically competed for AhR binding with a DRE-containing oligonucleotide from the mouse CYP1A1 gene. The results suggest a possible mechanism by which TCDD could be acting to modulate ER levels.

Animals↗

PubMed1992

Chelatable metal ions are not required for aryl hydrocarbon receptor transformation to a DNA binding form: phenanthrolines are possible competitive antagonists of 2,3,7,8-tetrachlorodibenzo-p-dioxin.

The aryl hydrocarbon receptor (AhR) mediates the toxicity of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and related compounds by binding DNA and altering gene transcription. We determined whether AhR transformation to a DNA binding form requires chelatable metal ions. The chelator 1,10-phenanthroline and its nonchelating isomers 1,7- and 4,7-phenanthroline blocked, in a concentration-dependent manner, TCDD-elicited transformation of the AhR in rat hepatic cytosol to a form which bound a dioxin-response element (DRE; upstream of the structural gene for cytochrome P4501A1). This was found to be due to the ability of these compounds to competitively inhibit [3H]TCDD specific binding to the AhR under conditions in vitro. EDTA (20 mM) failed to inhibit DRE binding of the transformed AhR, but pretreatment of cytosol with EDTA prior to transformation inhibited DRE binding up to 60%. However, removal of EDTA from the cytosol by gel filtration prior to incubation with TCDD resulted in the same DRE binding as filtered control cytosol without the added divalent metal ions. Both chelators, oxalic acid and iminodiacetic acid, failed to inhibit DRE binding when added prior to AhR transformation. Together these data indicate that chelatable metal ions are not required for AhR transformation to the DNA binding form.

Animals↗

PubMed1991

Ribonuclease inhibits Ah receptor transformation in vitro.

The aryl hydrocarbon (Ah) receptor undergoes a ligand-dependent transformation to a heteromeric structure which has the ability to bind DNA sequence-specifically with high affinity. By this mechanism, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and related xenobiotics modify gene expression. We observed that transformation was inhibited in vitro by the presence of ribonuclease A (RNAase) during incubation of rat hepatic cytosol with TCDD. This effect was detected as a decreased ability of the TCDD-receptor complex to bind to calf thymus DNA covalently linked to Sepharose, and to a dioxin-responsive enhancer which is upstream of the cytochrome P450IA1 structural gene. RNAase had no effect on previously transformed TCDD-receptor complexes. These observations indicated that RNAase acted during ligand binding and/or transformation of the Ah receptor. Saturation binding analyses demonstrated that RNAase decreased the receptor affinity for TCDD without changing the total number of binding sites. RNAase also inhibited transformation of the TCDD-bound, partially purified, untransformed, receptor. Thus RNAase does not interfere with ligand binding, but inhibits the subsequent transformation of the receptor monomer to the heteromeric, transcriptionally active, form.

Animals↗

PubMed1991

Characterization of multiple forms of the Ah receptor: recognition of a dioxin-responsive enhancer involves heteromer formation.

We have employed a combination of gel retardation, protein-DNA cross-linking, and protein-protein cross-linking techniques to further examine the 2,3,7,8-tetrachlorodibenzo-p- dioxin-(TCDD-) dependent changes in the Ah receptor that result in a DNA-binding conformation. Gel retardation analysis of DNA-Sepharose chromatographic fractions of rat hepatic cytosol indicated that TCDD-dependent and sequence-specific DNA binding coeluted with a 200-kDa form of the Ah receptor (peak 2) previously characterized as being multimeric and having high affinity for calf thymus DNA. The TCDD-bound, 100-kDa form of the receptor (peak 1) bound weakly to the DNA recognition motif. These results indicated that the DNA-binding form of the Ah receptor is a multimer. SDS-polyacrylamide gel electrophoresis of peak 2 cross-linked to a bromodeoxyuridine-substituted DNA recognition motif indicated that this form of the receptor present in rat hepatic cytosol is composed of at least two DNA-binding proteins of approximately 100 and 110 kDa. Using the chemical cross-linking agent dimethyl pimelimidate, we further established that the 100-kDa form of the receptor (peak 1) associates with a different protein to generate the receptor form (peak 2) that binds to the dioxin-responsive enhancer. Photoaffinity-labeling studies indicated that only the 100-kDa protein (peak 1), and not the 110-kDa protein, binds ligand. Together, these observations imply that the DNA-binding form of the Ah receptor exists as a heteromer.

Affinity Labels↗

PubMed1991

Determination of individual porphyrins in rodent urine using high-performance liquid chromatography following clean-up by anion-exchange chromatography.

We describe a method for the rapid clean-up of rodent urine samples prior to the analysis of porphyrin carboxylic acids by reversed-phase high-performance liquid chromatography (HPLC) with fluorescence detection. A simple pretreatment step using chromatography on a Dowex 1X8 anion-exchange resin effectively removes fluorescent substances that are present in rodent urine and would otherwise interfere with the detection and quantitation of urinary porphyrins by HPLC. Recovery of porphyrins with four to eight carboxyl groups (coproporphyrin to uroporphyrin) averaged 93% using this procedure. The use of this method to determine the amount of individual porphyrins present in the urine of hexachlorobenzene-treated mice is illustrated.

Animals↗

PubMed1991

Inhibition and reconstitution of Ah receptor transformation in vitro: role and partial characterization of a cytosolic factor(s).

The Ah receptor binds 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and related aryl hydrocarbons and mediates their biochemical and toxic effects by modifying gene expression. In order to interact with DNA, the TCDD.receptor complex must undergo a poorly understood transformation to a form which is distinguishable by its increased affinity for DNA-Sepharose and for its specific enhancer element upstream from the cytochrome P450IA1 gene. We have found that this transformation process is inhibited in vitro by treatment of rat hepatic cytosol with activated charcoal prior to addition of [3H]TCDD. The transformed form of the receptor can be generated in the charcoal-inhibited cytosol by incubation with hepatic cytosol from either DBA/2J mice (in which [3H]TCDD-specific binding is undetectable under these conditions) or rat (in which Ah receptor was prebound with unlabeled ligand). Transformation is observed whether this addition occurs before or after [3H]TCDD is bound to the charcoal-treated receptor. Thus, transformation is (i) mediated by some additional cytosolic component(s) and (ii) separable from ligand binding. The untransformed [3H]TCDD.receptor complex, isolated by DNA-Sepharose chromatography, can also be transformed if DBA mouse hepatic cytosol is added. This partially purified untransformed receptor preparation and gel retardation analysis were used to further characterize the transforming activity in DBA cytosol. We observed that the "Ah receptor transforming factor" is heat-labile, trypsin-sensitive, removed or inactivated by charcoal, of greater than approximately 50 kDa, and elutes from Superose at a Rs of approximately 6 nm. In conjunction with our previous studies documenting the increased molecular weight of the transformed compared to the untransformed Ah receptor, and identifying the heteromeric structure of the transformed receptor, we postulate that the ligand-binding subunit (the untransformed receptor) must associate tightly with another cytosolic protein, which is also present in the receptor-defective DBA mouse, in order to transform to the transcriptionally active receptor.

Animals↗

PubMed1991

Alpha-naphthoflavone acts as an antagonist of 2,3,7, 8-tetrachlorodibenzo-p-dioxin by forming an inactive complex with the Ah receptor.

alpha-Naphthoflavone (ANF) has previously been shown to compete with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) for binding to the Ah receptor under conditions in vitro. However, ANF also prevents TCDD-elicited cytochrome P450lA1 induction, immunosuppression, and down-regulation of the estrogen receptor in vivo and within intact isolated cells. These data suggest that ANF is a TCDD antagonist. This study investigated the ability of ANF to transform the Ah receptor contained in rat hepatic cytosol or mouse hepatoma cells to a form that recognizes the dioxin-responsive enhancer element (DRE) upstream of the cytochrome P450lA1 gene. Gel retardation analysis indicated that TCDD- or beta-naphthoflavone (BNF)-bound receptor was able to bind to the DRE, whereas essentially no receptor-DRE complexes were observed using cytosol incubated with ANF concentrations as high as 1000 nM. Furthermore, an excess of ANF, when added to cytosol just before TCDD, blocked, in a concentration-dependent manner, the ability of TCDD to transform the receptor to a form that bound to the DRE. These studies indicated that ANF binds to the receptor and confers on it a conformation that cannot recognize the DNA recognition sequence contained in the DRE. Although an excess of the agonist 2,3,7,8-tetrachlorodibenzofuran (TCDF) readily reversed the inhibitory actions of ANF, ANF was unable to reverse the effects of TCDD, TCDF, or BNF on the receptor. These studies suggested that TCDD binding, unlike that of ANF, results in a receptor conformation that has higher affinity for the ligand. Treatment of mouse hepatoma Hepa 1c1c7 cells with TCDD or BNF resulted in receptor contained in nuclear extracts that bound to the DRE. Only a very minor ligand-dependent protein-DNA complex was detected when cells were treated with ANF. These data indicated that ANF acts as an antagonist of TCDD by directly binding to the Ah receptor and eliciting a protein conformation that has very low affinity for DNA.

Animals↗

PubMed1990

Protein-DNA interactions at a dioxin-responsive enhancer. Evidence that the transformed Ah receptor is heteromeric.

The Ah receptor in rat hepatic cytosol was transformed to a DNA-binding form by incubation in vitro with the ligand 2,3,7,8-tetrachlorodibenzo-p-dioxin. The transformed receptor was covalently cross-linked to a bromodeoxyuridine-substituted DNA recognition motif by exposure to ultraviolet irradiation. Analyses of the cross-linked protein-DNA complexes by gel electrophoresis and autoradiography imply that the DNA-binding form of the liganded Ah receptor is composed of two protein components, whose molecular masses are about 110 and 100 kDa. Protease digestion studies suggest that the two components have different primary structures. Photoaffinity labeling studies imply that the smaller protein is the ligand-binding component of the receptor. These findings constitute biochemical evidence that the DNA-binding form of the Ah receptor is a heterodimer.

Animals↗

PubMed1990

Impairment of prothymocyte activity by 2,3,7,8-tetrachlorodibenzo-p-dioxin.

Exposure of experimental animals to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) results in severe thymic atrophy and suppression of cell-mediated and humoral immune functions. However, despite much effort the mechanism by which TCDD produces these responses, particularly thymic atrophy, remains unclear. In this report, we have examined the effect of acute TCDD exposure on lymphocyte stem cells in young adult BALB/c mice to determine whether alterations to events early in T lymphopoiesis contribute to TCDD-induced thymic atrophy. TCDD produced a dose-dependent reduction in thymic weight and cellularity following a single dose of 5 to 120 micrograms TCDD/kg. This thymic atrophy correlated with a dose-dependent suppression of the biosynthesis and mRNA levels of the lymphocyte stem cell-specific DNA polymerase terminal deoxynucleotidyl transferase in bone marrow and thymus. However, the reduction in thymic terminal deoxynucleotidyl nucleotidyl transferase synthesis, on a per cell basis, was less than that observed in bone marrow. Intrathymic CD4/CD8 and IL-2R expression demonstrated only mild alterations after exposure to 30 micrograms TCDD/kg. These data suggest that thymocytes are more refractory to TCDD than are pre-T cells. To assess this possibility directly, bone marrow prothymocytes from TCDD-treated donor mice were examined for their capacity to reconstitute the thymuses of adoptive, irradiated recipients. Our results indicate that prothymocyte activity was severely impaired by TCDD exposure and that this effect occurred at low tissue levels of TCDD. In contrast, we observed no reduction in the number of colony-forming unit-granulocyte macrophage and a moderate decrease in colony-forming unit-spleen. These data suggest that TCDD-induced thymic atrophy is the result, at least in part, of impaired thymic seeding by prothymocytes.

Animals↗

PubMed1990

Human body burden of polychlorinated dibenzofurans associated with toxicity based on the yusho and yucheng incidents.

The polychlorinated dibenzofurans (PCDFs) are one group of man-made toxicants for which reasonably extensive data exist relevant to dose-response relationships in humans. Examination of contaminated food oil consumption from the yusho (Japan) poisoning incident indicates the mean uptake or body burden of 2, 3, 4, 7, 8-pentachlorodibenzofuran (PnCDF) equivalents (PEQ) associated with nausea and anorexia to be 4.4 micrograms/kg body wt and that associated with chloracne to be 5.9 micrograms/kg. For the yucheng (Taiwan) poisoning incident, blood measurements for chloracne show a similar body burden of 4.0 micrograms/kg. The latter value is toxicologically equivalent to a 2,3,7,8-tetrachlorodibenzo-p-dioxin equivalent (TEQ) body burden of 2.0 micrograms/kg body wt or about 150 micrograms for an adult person. This corresponds to an adipose tissue level of about 10 micrograms/kg fat, and is comparable to that known to cause chloracne in rhesus monkeys. These body burdens on a TEQ basis are more than 200 times higher than the average current levels of PCDDs/PCDFs found in North American populations and are the first to relate human body burdens of PCDFs with a known effect and to compare them to animal data. Since the effects reported may not be the most sensitive indicator of human toxicity, lower body burdens could be associated with more subtle toxicological events.

Adolescent↗

PubMed1990

Prothymocyte activity is reduced by perinatal 2,3,7,8-tetrachlorodibenzo-p-dioxin exposure.

The mechanism by which exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) produces thymic atrophy and cell-mediated immune suppression in experimental animals is poorly understood. A previous study from our laboratory found that terminal deoxynucleotidyl transferase-synthesizing lymphocyte stem cell populations in fetal liver and neonatal bone marrow, but not thymus, were profoundly altered after perinatal TCDD exposure, implying that a defect in the prothymocyte population in liver and marrow may play a role in the etiology of thymic atrophy in TCDD-exposed animals. In this report, we present results of experiments designed to directly assess the prothymocyte compartment in mice exposed to TCDD perinatally by examining the ability of these stem cells to reconstitute an irradiated thymus. Maternal TCDD exposure (15 micrograms/kg) caused a significant impairment of both fetal liver and neonatal bone marrow prothymocyte activity. These alterations occurred at tissue concentrations less than 200 fg of TCDD per mg. TCDD treatment also resulted in a mild reduction in colony-forming unit-spleen in these organs and a decrease in colony-forming unit-granulocyte-macrophage in fetal and neonatal liver, but not bone marrow. Overall, these data provide evidence that alterations to early stages of T-lymphopoiesis, at the level of the prothymocyte, may be involved in the development of TCDD-induced thymic atrophy and cell-mediated immunosuppression.

Animals↗

PubMed1989

Characterization of multiple forms of the Ah receptor: comparison of species and tissues.

Biochemical and toxic responses to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) appear to be mediated via the Ah receptor, a gene-regulatory protein that, like steroid hormone receptors, undergoes a ligand-dependent acquisition of affinity for nuclei and DNA. Since responses to TCDD are highly species- and tissue-specific, we compared DNA-binding properties of Ah receptor from several tissues of rat, C57BL/6 mouse, hamster, and guinea pig, using DNA-Sepharose chromatography. Hepatic cytosol from all species contained TCDD.receptor complexes that eluted at approximately 0.15 (peak 1) and approximately 0.33 M NaCl (peak 2). The relative proportions of these forms as well as of TCDD-receptor that did not bind to DNA (i.e., was present in flowthrough fractions) varied among species. In each case, the yield of the higher affinity form (peak 2) increased with time or temperature of incubation. Cytosol from lung, thymus, kidney, and testis contained the same two forms; peak 2 was the major DNA-binding form only in thymus. In KCl extracts of hepatic nuclei from animals treated with [3H]TCDD, only the higher affinity form (peak 2) was found. Peak 1 isolated from cytosol by DNA-Sepharose and incubated with hepatic cytosol from D2 mouse (which contains no detectable receptor) transformed into peak 2, suggesting that these two forms are different conformations of the same protein. Sucrose density gradient and gel filtration analyses of peaks 1 and 2 isolated from DNA-Sepharose indicated that (i) the untransformed form (peak 1) was smaller than the unoccupied and the transformed forms, (ii) 0.4 M KCl in the density gradients had little effect on these isolated forms, and (iii) nuclear receptor sedimented like peak 2. On the basis of these results, we hypothesize that the Ah receptor exists in several forms: When occupied, it has no affinity for DNA. Ligand binding initially yields a smaller form with low DNA affinity (i.e., peak 1), as well as, in some cases, a form with no DNA affinity (flowthrough fractions); further incubation in the presence of cytosolic factor(s) induces a change conferring higher DNA affinity and faster sedimentation (i.e., peak 2). The latter form is likely the transcriptionally active form in vivo. Species and tissue differences in this scheme are quantitative rather than qualitative.

Animals↗

PubMed1989

Dioxins and the Ah receptor.

Despite continuing controversies related to public policy, information on the molecular biology of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) has advanced significantly over the past decade. Current understanding of the biological mechanisms of TCDD action is based upon the interactions of TCDD with a genetically expressed cytosolic macromolecule that functions as a receptor in many cells across many species. The Ah receptor recognizes TCDD and structurally similar molecules and serves as the transducing step whereby TCDD alters gene expression through the association of the TCDD:receptor complex with specific TCDD-responsive elements on the genome. Understanding these molecular events and their relevance to the organ-level manifestations of TCDD toxicity may be critical to formulating scientifically based assessments of the risk of TCDD exposure.

Dioxins↗

PubMed1989

Interaction of hexachlorobenzene with the receptor for 2,3,7,8-tetrachlorodibenzo-p-dioxin in vitro and in vivo. Evidence that hexachlorobenzene is a weak Ah receptor agonist.

Hexachlorobenzene (HCB) produces hepatic porphyria and induces the hepatic cytochrome P450 isozymes P450c (P450IA1) and P450d (P450IA2) in rodents. These and other effects of HCB resemble those of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), which acts via its binding to the aromatic hydrocarbon (Ah) receptor. We therefore examined the ability of HCB to interact with this receptor in vitro and in vivo. HCB, at concentrations of 1 microM or higher, inhibited the specific binding of [3H]TCDD (0.3 nM) to the Ah receptor in vitro, whereas the solubility of [3H]TCDD was affected only at 100 microM HCB. The inhibition was competitive, with a KI of approximately 2.1 microM. In rats fed a diet containing 3000 ppm HCB for varying times (4 h to 7 days), the specific binding of [3H]TCDD in hepatic cytosol was reduced by up to 40%, as observed previously for known Ah receptor agonists. The decrease in [3H]TCDD specific binding in cytosol of HCB-treated rats was due principally to a decrease in the number of binding sites for [3H]TCDD rather than competition from residual HCB. As shown by immunoblotting and radioimmunoassay, HCB induced the cytochrome P450 isozymes P450c and P450d, which are regulated by the Ah receptor, as well as the phenobarbital-inducible isozymes P450b and P450e. Together these results indicate that HCB is a weak agonist for the Ah receptor, and suggest that some of its effects may be mediated by its interaction with this gene-regulatory protein.

Animals↗