Biomedical subjects

S Safe

Publications and source records attributed to S Safe.

At least 181 records · Page 10Linked to original sources

PubMed1989

Photoaffinity labeling of the nuclear Ah receptor from mouse Hepa 1c1c7 cells using 2,3,7,8-[3H]tetrachlorodibenzo-p-dioxin.

The photoinduced formation of the covalently labeled cytosolic and nuclear aryl hydrocarbon (Ah) receptors was studied using 2,3,7,8-[3H]tetrachlorodibenzo-p-dioxin (TCDD) as the photoaffinity label. Irradiation of TCDD alone at wavelengths of greater than 300 nm resulted in rapid degradation of this compound (t 1/2 = 8 min). In a separate experiment, the unliganded cytosolic Ah receptor was only slowly inactivated (t 1/2 = 48 min) using the greater than 300 nm light source. Preliminary experiments with rat hepatic cytosol did not result in significant formation of specifically bound [3H]TCDD-protein covalent adducts which could be visualized by autoradiography. Irradiation of [3H]TCDD-nuclear Ah receptor complexes isolated from mouse Hepa 1c1c7 cells for 15 min gave approximately a 40% overall yield of the radiolabeled Ah receptor protein adduct. Denaturing sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the [3H]TCDD-nuclear Ah receptor photoadduct gave a single major radiolabeled protein with an apparent molecular size of 91 kDa. The chromatographic properties of the control (dark) and photolabeled nuclear Ah receptor complexes were comparable using Sephacryl S-300 and DNA-Sepharose columns. Velocity sedimentation of both the control (dark) and irradiated nuclear Ah receptor complexes gave specifically bound peaks which sedimented at 6.5 S. However, the trichloroacetic acid-precipitable (buffer-reconstituted) [3H]TCDD-nuclear Ah receptor photo-covalent adduct was eluted from the Sephacryl S-300 column in the void volume and did not exhibit a specifically bound peak after velocity sedimentation analysis due to protein aggregate formation. In contrast, the elution profile of the aggregate on a DNA-Sepharose column was similar to that observed for the control (dark) and photolabeled complexes, which were eluted from the column with salt concentrations between 0.24 and 0.28 M. These photolabeling studies show that [3H] TCDD can act as a photoaffinity label for the Ah receptor and can be utilized as photoligand to probe further the structure and function of this protein.

Affinity Labels↗

PubMed1989

2,3,7,8-Tetrachlorodibenzo-p-dioxin-induced porphyria in genetically inbred mice: partial antagonism and mechanistic studies.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) (233 nmol/kg) causes a significant increase of hepatic uroporphyrin, heptacarboxyporphyrin, and total porphyrins in female C57BL/6 mice, ovariectomized C57BL/6 mice, male C57BL/10 mice, and male C57BL/6 mice 3 weeks after treatment. In contrast, 6-methyl-1,3,8-trichlorodibenzofuran (MCDF) was inactive at a dose of 750 mumol/kg. Cotreatment of the mice with TCDD (233 mol/kg) plus MCDF (750 mumol/kg) resulted in partial antagonism of TCDD-induced hepatic porphyrin accumulation only in the female mice. Parallel studies in female C57BL/6 mice showed that the TCDD-induced porphyria was accompanied by the induction of hepatic microsomal aryl hydrocarbon hydroxylase (AHH) and ethoxyresorufin O-deethylase (EROD) activities and the depression of uroporphyrinogen decarboxylase (UROD). MCDF (750 mumol/kg) did not significantly affect these enzymes. In the cotreatment studies (MCDF plus TCDD), MCDF partially antagonized TCDD-induced hepatic porphyrin accumulation but did not affect the levels of hepatic AHH, EROD, or UROD. These results indicate that other factors, in addition to the induction of cytochrome P450-dependent monooxygenases and depressed UROD activity, are important in TCDD-induced porphyria in C57BL/6 female mice.

Animals↗

PubMed1989

Structure-dependent induction of aryl hydrocarbon hydroxylase in human breast cancer cell lines and characterization of the Ah receptor.

The structure-dependent induction of aryl hydrocarbon hydroxylase (AHH) and ethoxyresorufin O-deethylase by 2,3,7,8-tetrachlorodibenzo-p-dioxin, 2,3,7,8-tetrachlorodibenzofuran, and 1,2,4,7,8-pentachlorodibenzo-p-dioxin was determined in the MCF-7, T47-D, and MDA-MB-231 human breast cancer cell lines. Both the MCF-7 and T47-D cells were responsive to the induction effects of the halogenated aryl hydrocarbons and the structure-induction relationships were comparable to the reported structure-activity (induction, receptor binding, and toxicity) relationships observed in rodents and rodent cells in culture. The induction of ethoxyresorufin O-deethylase in the T47-D cells was the most sensitive aryl hydrocarbon (Ah) receptor-mediated response in both cell lines and this enzyme activity was more inducible than aryl hydrocarbon hydroxylase. In contrast, the three congeners were inactive as monooxygenase enzyme inducers in the MDA-MB-231 cells. Despite the differential Ah responsiveness of the cell lines, incubation of the cells with [3H]-2,3,7,8-tetrachlorodibenzo-p-dioxin followed by extraction of the nuclei with high salt and velocity sedimentation analysis of the extracts showed that specifically bound nuclear Ah receptor complexes were present in the three cell lines. The sedimentation coefficients (and levels) for the nuclear receptors were 6.6 S (32.1 fmol/mg protein/mg DNA), 6.9 S (61.6 fmol/mg protein/mg DNA), and 7.4 S (38.2 fmol/mg protein/mg DNA) in the T47-D, MCF-7, and MDA-MB-231 cell lines, respectively. Cytosolic receptor was also detected in the MCF-7 and MDA-MB-231 cells. Thus, despite the differences in Ah responsiveness of the T47-D and MDA-MB-231 cells, comparable levels of nuclear receptor were detected in both cell lines. Furthermore, the elution profiles of the nuclear receptors from DNA-Sepharose columns by using a salt gradient were similar and this suggested that defects in the DNA-binding activity of MDA-MB-231 nuclear receptor complexes were not major factors associated with their failure to respond to 2,3,7,8-tetrachlorodibenzo-p-dioxin and related compounds.

Aryl Hydrocarbon Hydroxylases↗

PubMed1989

Induction of cytochrome P450-dependent monooxygenase activities in rat hepatoma H-4-IIE cells in culture by 2,3,7,8-tetrachlorodibenzo-p-dioxin and related compounds: mechanistic studies using radiolabeled congeners.

Treatment of rat hepatoma H-4-IIE cells in culture with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), 2,3,7,8-tetrachlorodibenzofuran (TCDF), 1,2,3,7,8-pentachlorodibenzo-p-dioxin (PeCDD), 1,2,3,7,8-pentachlorodibenzofuran (PeCDF), 1,2,7,8-TCDF, and 2,3,7-trichlorodibenzo-p-dioxin (TrCDD) resulted in the structure-dependent induction of aryl hydrocarbon hydroxylase and ethoxyresorufin O-deethylase activities. The induction potencies followed the order 2,3,7,8-TCDD greater than 2,3,7,8-TCDF greater than 1,2,3,7,8-PeCDD approximately 1,2,3,7,8-PeCDF greater than 1,2,7,8-TCDF greater than 2,3,7-TrCDD and were comparable to structure-toxicity relationships which have previously been reported. In contrast, many of the properties of these compounds were structure-independent. For example, using tritiated congeners of high specific activity (greater than 30 Ci/mmol) the sedimentation coefficients (S) for the nuclear and cytosolic aryl hydrocarbon (Ah) receptor complexes were 5-6 and 9-10 S, respectively, for all the radioligands. Moreover, examination of the processing of nuclear Ah receptor complexes for the radiolabeled congeners showed that after 6 h, the rates of nuclear processing were very low and varied between 0.006 and 0.0385 fmol degraded/mg protein/mg total DNA. These results were consistent with the reported stability and persistence of the nuclear Ah receptor complexes and in addition, there were no apparent structure-dependent differences in the processing rates. Inspection of the nuclear receptor levels and the corresponding induced enzyme activities for the congeners showed that there was a linear correlation between average nuclear receptor complex levels (18-42 h) and induced enzyme activities (32-42 h) for all six radioligands; these data indicated that the rates of cytochrome P450-dependent gene expression correlated with the levels of nuclear Ah receptor complex. In contrast, the accumulation of occupied nuclear receptor complexes in rat hepatoma H-4-IIE cells was structure-dependent and appeared to be one of the factors which governed the observed structure-induction and the previously reported structure-toxicity relationships for 2,3,7,8-TCDD and related halogenated aryl hydrocarbons.

Animals↗

PubMed1989

2,2',4,4',5,5'-hexachlorobiphenyl as a 2,3,7,8-tetrachlorodibenzo-p-dioxin antagonist in C57BL/6J mice.

At doses as high as 750 to 1000 mumol/kg, 2,2',4,4',5,5'-hexachlorobiphenyl (HCBP) did not cause fetal cleft palate, suppress the splenic plaque-forming cell response to sheep red blood cells, or induce hepatic microsomal ethoxyresorufin O-deethylase (EROD) in C57BL/6J mice. Despite the lack of activity of HCBP in eliciting any of these aryl hydrocarbon (Ah) receptor-mediated responses, competitive binding studies indicated that HCBP competitively displaced 2,3,7,8-[3H]tetrachlorodibenzo-p-dioxin (TCDD) from the murine hepatic cytosolic receptor. Cotreatment of C57BL/6J mice with TCDD (3.7 nmol/kg) and HCBP or 4,4'-diiodo-2,2',5,5'-tetrachlorobiphenyl (I2-TCBP) (400 or 1000 mumol/kg) showed that both compounds partially antagonized TCDD-mediated cleft palate and immunotoxicity (i.e., suppression of the splenic plaque-forming cell response to sheep red blood cells), and HCBP antagonized TCDD-mediated hepatic microsomal EROD induction. Thus, HCBP and I2-TCBP, like the commercial polychlorinated biphenyl mixture Aroclor 1254, were partial antagonists of TCDD action in C57BL/6J mice; however, it was also apparent from the results that Aroclor 1254 was the more effective antagonist at lower doses. Using [3H]TCDD, it was also shown that some of the effects of HCBP on TCDD-mediated cleft palate may be due to the decreased levels of TCDD found in the fetal palates after cotreatment with TCDD and HCBP. 4,4'-[125I2]diiodo-2,2',5,5'-tetrachlorobiphenyl ([125I2]TCBP) of high specific activity (3350 Ci/mmol) was synthesized and used to investigate the direct binding of this compound to the murine hepatic Ah receptor or other cytosolic proteins. No direct specific binding was observed between 125I2-TCBP and any cytosolic proteins using a sucrose density gradient assay procedure. These results contrasted with previous studies with Aroclor 1254 that suggested that this mixture acted as a competitive Ah receptor antagonist.

Animals↗

PubMed1989

Polychlorinated biphenyls (PCBs): mutagenicity and carcinogenicity.

The potential mutagenicity and carcinogenicity of commercial PCBs has been investigated in both in vivo and in vitro systems and several conclusions can be drawn from these studies. (1) PCBs can covalently adduct DNA both in vivo and in vitro (using a source of metabolic activation); the more highly chlorinated biphenyls are poorly metabolized and these compounds tend to exhibit very low binding to DNA. Based on the structure-activity relationships for PCBs (Safe, 1984) it is unlikely that the more toxic compounds such as 3,3',4,4',5-penta- and 3,3',4,4',5,5'-hexachlorobiphenyl, would form covalent adducts with DNA. (2) PCB mixtures and individual compounds exhibit minimal mutagenic activity in most assay systems. (3) The more highly chlorinated PCB mixtures (i.e. greater than 50% Cl by weight) are hepatocarcinogens in rodents whereas data from a limited number of studies suggest that the lower chlorinated mixtures are not carcinogenic. (4) In some model systems, the higher chlorinated PCB mixtures act as promoters of preneoplastic lesions and hepatocellular carcinomas in rodents treated with a variety of initiators. (5) Aroclor 1254 acts as a promoter of skin papilloma formation in HRS/J hairless mice and structure-activity and genetic studies suggest that the Ah receptor is necessary but not sufficient for the activity of halogenated aryl hydrocarbons as promoters in hairless mice. (6) Individual PCB congeners and higher chlorinated commercial mixtures also exhibit anti-carcinogenic activity in the CD-1 mouse skin cancer model. (7) Results from occupational studies suggest that individuals exposed to PCBs may have an excess of cancer at some sites, however, the most comprehensive study (Brown, 1987) suggests that there are no significant increases in the overall cancer rate in workers exposed to PCBs. Follow-up and continuing epidemiological studies on the PCB-exposed workers are required to further clarify the potential carcinogenic effects of PCBs on humans. In several strains of rats and mice, there is a high incidence of hepatic preneoplastic lesions and carcinomas and these lesions can be induced by diverse promoting agents (Schulte-Hermann et al., 1983; Weinstein, 1984). Since PCBs are not mutagenic and do not readily form covalent adducts with cellular DNA, it is likely that the higher chlorinated biphenyls are not genotoxic and act as promoters of carcinogenesis in rodents. A comparable mechanism has been suggested for 2,3,7,8-TCDD (Shu et al., 1987; Weinstein, 1984). For PCBs, the role of the Ah receptor in mediating their activity as promoters has not been delineated.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗

PubMed1989

6-Methyl-1,3,8-trichlorodibenzofuran (MCDF) as a 2,3,7,8-tetrachlorodibenzo-p-dioxin antagonist in C57BL/6 mice.

6-Methyl-1,3,8-trichlorodibenzofuran (MCDF), 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and TCDD plus MCDF were administered to C57BL/6 mice and their effects on several aryl hydrocarbon (Ah) receptor-mediated responses including hepatic microsomal aryl hydrocarbon hydroxylase (AHH) and ethoxyresorufin O-deethylase (EROD) induction, immunotoxicity and teratogenicity were determined. MCDF did not induce hepatic microsomal AHH and EROD at doses up to 500 mumol/kg, however, co-administration of MCDF (50 mumol/kg) with a dose of TCDD which elicited a submaximal induction response (i.e. ED80-100, 15 nmol/kg) resulted in some small but significant inhibition of the induction of hepatic microsomal AHH and EROD (14 and 17%, respectively) compared to that observed with TCDD alone. Co-administration of TCDD and other doses of MCDF (10, 100, 200 or 500 mumol/kg) did not effect the induction response. These results were in contrast to the effectiveness of MCDF as an antagonist of the induction of AHH and EROD by TCDD in the rat (up to 50% inhibition of monooxygenase induction). Administration of MCDF (4, 20 and 40 mumol/kg) to C57BL/6 mice caused some inhibition of the splenic plaque-forming cell response to sheep erythrocytes only at the highest dose (26% decrease); the interaction of MCDF (4, 20 and 40 mumol/kg) and an immunotoxic dose of TCDD (3.7 nmol/kg) resulted in significant protection from the immunotoxic effects of TCDD at the 2 higher dose levels of MCDF. Similarly, MCDF (400 mumol/kg) did not cause cleft palate in mice but at this dose level MCDF afforded some protection from TCDD (20 micrograms/kg)-mediated cleft palate in mice. However, studies utilizing [3H]TCDD suggested that the protective effects may be due to modulation of TCDD reaching the palate in the co-treated animals (MCDF plus TCDD). Although both MCDF and Aroclor 1254 were both weak Ah receptor agonists in C57BL/6 mice, the former compound was much less effective as a TCDD antagonist. The observed species-specific effects for these 2 TCDD antagonists may be related species-dependent differences in receptor structure and receptor-ligand (i.e. agonist or antagonist) interactions.

Abnormalities, Drug-Induced↗

PubMed1989

Dose-response immunotoxicities of commercial polychlorinated biphenyls (PCBs) and their interaction with 2,3,7,8-tetrachlorodibenzo-p-dioxin.

The relative potencies of the commercial polychlorinated biphenyl (PCB) mixtures Aroclors 1260, 1254, 1248, 1242, 1016 and 1232 to inhibit the murine splenic plaque-forming cell response to sheep red blood cells was determined by dose-response treatment of C57BL/6 mice followed by logit plot analysis of the data. The ED50 values obtained for Aroclors 1260, 1254, 1248, 1242, 1016 and 1232 were 104, 118, 190, 391, 408 and 464 mg/kg or 0.28, 0.35, 0.66, 1.5, 1.5 and 2.0 mmol/kg, respectively. It was apparent that the higher chlorinated PCB preparations (Aroclors 1260, 1254 and 1248) were more potent than the lower chlorinated preparations (Aroclors 1242, 1016 and 1232). Previous studies have shown that the interaction of a subeffective dose of Aroclor 1254 (25 mg/kg) with an immunotoxic dose (3.7 nmol/kg) of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) resulted in significant antagonism of the toxicity of the latter compound. Co-treatment of mice with a 25 mg/kg dose of all the commercial Aroclors and with a 50 mg/kg dose of a reconstituted PCB mixture (resembling a PCB extract from human milk) with TCDD (3.7 nmol/kg) showed that, with the exception of Aroclor 1232, all of the commercial PCBs and the reconstituted PCB mixture significantly antagonized the TCDD-mediated inhibition of the splenic plaque-forming cell response in C57BL/6 mice. The identities of the PCB congeners responsible for this antagonism and the mechanism of this process are unknown and are currently being investigated.

Animals↗

PubMed1989

Induction of glutathione S-transferases in genetically inbred male mice by dietary ethoxyquin hydrochloride.

1. Constitutive and ethoxyquin hydrochloride (EQ-HCl)-induced hepatic glutathione (GSH) S-transferase, GSH reductase, and GSH peroxidase activities were determined in 5 strains of 8-10 week old inbred male mice. 2. The constitutive GSH S-transferase (GST) activity varied from 2.9 (SJL/JCR) to 8.9 (C57BL/6NCR) mumol product formed/min/mg protein and the corresponding values for the EQ-HCl-treated mice were in the range of 15.3-25.3 mumol product formed/min/mg protein. 3. EQ-HCl induced GST activity in all the strains examined and this contrasted to the induction activity of Aroclor 1254 which was strain-dependent. GST activity was induced 2.9-fold in Aroclor 1254-responsive (C57BL/6) and 2.8-fold in non-responsive (DBA/2) mice, respectively.

Animals↗

PubMed1989

Development and validation of in vitro induction assays for toxic halogenated aromatic mixtures: a review.

Halogenated aromatic industrial compounds, typified by the polychlorinated dibenzo-p-dioxins (PCDDs), dibenzofurans (PCDFs) and biphenyls (PCBs) have been identified as residues in almost every component of the global ecosystem. Risk assessment of the complex mixtures of halogenated aromatics found in environmental samples is complicated by analytical problems and the lack of toxicological information on individual compounds and mixtures. Research in our laboratory has focused on the development and vadidation of the in vitro aryl hydrocarbon hydroxylase (AHH) induction assay in rat hepatoma H-4-II E cells in culture for quantitating individual toxic halogenated aryl hydrocarbons and their mixtures. For several PCB, PCDD, PCDF congeners, their mixed bromo/chloro analogs and reconstituted mixtures there was an excellent linear correlation between their -log ED50 values for AHH induction in rat hepatoma cells and their -log ED50 values for in vivo hepatic microsomal AHH induction, inhibition of body weight gain and thymic atrophy in the rat. It has also been shown for selected compounds that there was a good correlation between their in vitro AHH induction potencies and their effects in guinea pigs (AHH induction, inhibition of body weight gain) and mice (immunotoxicity). This assay system has been utilized to quantitative the "2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) equivalents" present in extracts from diverse sources including fly ash from a municipal incinerator and pyrolyzed brominated flame retardants which contain a complex mixture of halogenated dibenzo-p-dioxins and dibenzofurans.

Animals↗

PubMed1989

Partial antagonism of 2,3,7,8-tetrachlorodibenzo-p-dioxin-mediated induction of aryl hydrocarbon hydroxylase by 6-methyl-1,3,8-trichlorodibenzofuran: mechanistic studies.

6-Methyl-1,3,8-trichlorodibenzofuran (MCDF) binds with moderate affinity to the aryl hydrocarbon (Ah) receptor protein (4.9 x 10(-8) M) but is a weak Ah receptor agonist. Cotreatment of male Long Evans rats with MCDF (50 mumol/kg) and a dose of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) that causes a near-maximal induction of hepatic microsomal aryl hydrocarbon hydroxylase and ethoxyresorufin O-deethylase activities resulted in a significant inhibition of these activities for up to 96 hr. Comparable results were obtained with MCDF (10(-7) M) and TCDD (10(-8) M) in rat hepatoma H-4-II E cells in culture over 36 hr. TCDD treatment of rats resulted in an initial decrease of hepatic cytosolic Ah receptor within 6 hr and this was followed by a subsequent 138% increase in cytosolic receptor levels 72 hr after treatment. Although MCDF (50 mumol/kg) did not significantly alter rat hepatic cytosolic Ah receptor levels in animals cotreated with TCDD plus MCDF, the latter compound significantly inhibited TCDD-mediated replenishment of the cytosolic Ah receptor. In contrast, treatment of rat hepatoma H-4-II E cells with TCDD (10(-8) M) resulted in the rapid (within 1 hr) depletion of cytosolic Ah receptor, which remained undetectable for up to 36 hr; cotreatment of the cells with MCDF (10(-7) M) and TCDD (10(-8) M) resulted in cytosolic Ah receptor levels that were similar to those observed after treatment with TCDD alone. The effects of MCDF on the uptake and persistence of nuclear [3H]TCDD-Ah receptor complex levels were also determined in rat liver and rat hepatoma H-4-II E cells in culture. MCDF did not significantly decrease levels of occupied nuclear Ah receptor complexes in the rat or rat hepatoma cells. Moreover, using the sucrose density gradient assay procedure, the sedimentation coefficients of the cytosolic and nuclear TCDD-Ah receptor complexes in the presence or absence of MCDF were comparable. The results of these and other related studies with 6-substituted-1,3,8-trichlorodibenzofurans suggest that MCDF may act as a partial TCDD antagonist by competing with TCDD for nuclear binding sites.

Animals↗

PubMed1988

Induction of rat liver microsomal cytochrome P-450 isozymes and epoxide hydrolase by a series of 4'-substituted-2,3,4,5-tetrachlorobiphenyls.

We have shown previously that 2,3,4,5-tetrachlorobiphenyl is ineffective as an inducer of rat liver microsomal cytochrome P-450. Addition of a single para-chloro substituent in the otherwise unsubstituted phenyl ring, to give 2,3,4,4',5-pentachlorobiphenyl, produces a potent cytochrome P-450 inducer with both phenobarbital- and 3-methylcholanthrene-type characteristics. In the present study, 2,3,4,5-tetrachlorobiphenyl was substituted in the para(4') position with 12 other functional groups. The 4'-X-C12H5Cl4 derivatives were tested as inducers of cytochromes P-450a--P-450e and epoxide hydrolase, by immunochemical analysis of liver microsomes prepared 4 days after a single treatment (500 mumol/kg) of 1-month-old male Long Evans rats. When the para' substituent was a halogen (F, Cl, Br or I), the derivative induced both cytochromes P-450b and P-450e, and cytochromes P-450c and P-450d, which are the major phenobarbital- and 3-methylcholanthrene-inducible isozymes, respectively. A similar type of induction was observed with a second group of derivatives substituted with CN, NO2 or CF3. However, a derivative containing CH3CO--(which is also a meta-directing, ring-activating substituent) failed to induce cytochromes P-450a-P-450e at the dosage and time tested. Members of a third group of derivatives, which contained an ortho/para-directing, ring-activating substituent) were either ineffective inducers (OH, CH3, CH3O--), or were inducers of cytochromes P-450c and P-450d (isopropyl or t-butyl). Hence, 4'-substitution with a bulky lipophilic substituent conferred 3-methylcholanthrene- but not phenobarbital-type characteristics on 2,3,4,5-tetrachlorobiphenyl. Some of the derivatives tested, namely those substituted with Cl, Br, I and CF3, were remarkably effective inducers of cytochrome P-450a, causing a 10-11-fold induction of this isozyme. Data on the induction of cytochrome P-450c were analyzed by multiparameter linear regression in an attempt to correlate the biological activity of the 4'-X-C12H5Cl4 derivatives with the physiochemical properties of the various substituents. From these results, and those reported recently, we propose that binding of the 4'-X-C12H5Cl4 derivatives to the rat cytosolic Ah receptor is favored by increasing the electronegativity, lipophilicity and hydrogen bonding characteristics of the 4' substituent, whereas enzyme induction (both in vivo and in cultured rat hepatoma cells) is also governed by a fourth characteristic, the STERIMOL factor, which gives a measure of the width of the substituent.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗

PubMed1988

Radioligand-dependent differences in the molecular properties of the mouse and rat hepatic aryl hydrocarbon receptor complexes.

A comparison of the molecular properties of the male Long-Evans rat and male C57BL/6 mouse hepatic cytosolic aryl hydrocarbon (Ah) receptor complex was determined using 2,3,7,8-[3H]tetrachlorodibenzo-p-dioxin (TCDD) and 2,3,7,8-[3H]tetrachlorodibenzofuran (TCDF) as radioligands. In low salt buffer, the sedimentation coefficients, Stokes radii, relative molecular masses, frictional ratios, axial ratios and gel permeation chromatographic properties of the rat receptor complexes were ligand independent. In contrast, there were several ligand-dependent differences in the mouse Ah receptor complexes formed after incubation in low salt buffer and these include: sucrose density gradient analysis of the 2,3,7,8-[3H]TCDF receptor complex gave a 9.5 S specifically bound peak and a 2.6 S nonspecifically bound peak whereas the corresponding 2,3,7,8-[3H]TCDD receptor complex gave a single 9.6 S specifically bound peak; sucrose density gradient analysis of the two major peaks eluted from a Sephacryl S-300 column chromatographic separation of the 2,3,7,8-[3H]TCDF receptor complex gave two specifically bound peaks at 9.2 and 5.1 S. The molecular properties of the rat hepatic cytosolic receptor complexes incubated in high salt (0.4 M KCl) buffer were ligand independent with one exception, namely the significant difference in the sedimentation coefficient of the specifically bound disaggregated 2,3,7,8-[3H]TCDD receptor complex (6.8 S) and the corresponding 2,3,7,8-[3H]TCDF receptor complex (5.0 S). The major ligand-dependent differences in the mouse receptor complexes incubated in high salt (0.4 M KCl) were associated with the sedimentation coefficients of the complexes derived after direct incubation and after gel permeation chromatography. For example, both ligands gave two specifically bound complexes after chromatography on Sephacryl S-300 column and centrifugation of these fractions gave both the approximately 9 and approximately 5 S peaks; this suggested that there was some equilibration between the aggregated and disaggregated receptor complexes. The behavior of the 2,3,7,8-[3H]TCDF mouse receptor complex was similar after incubation in low or high salt buffer except that sucrose density gradient analysis of the gel permeation chromatographic fractions gave an additional specifically bound peak which sedimented at 7.2 S. These studies demonstrate that the molecular properties of the Ah receptor were dependent on the source of the cytosolic receptor preparation, the ionic strength of the incubation media, and the structure of the radioligand.

Animals↗

PubMed1988

Comparative antiestrogenic activities of 2,3,7,8-tetrachlorodibenzo-p-dioxin and 6-methyl-1,3,8-trichlorodibenzofuran in the female rat.

The ED50s for the dose-response induction of hepatic microsomal aryl hydrocarbon hydroxylase (AHH) and ethoxyresorufin O-deethylase (EROD) by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in the female Sprague-Dawley rat were 3.3 and 2.7 nmol/kg, respectively. In contrast, the corresponding ED50 values for induction by the nontoxic 6-methyl-1,3,8-trichlorodibenzofuran (MCDF) were 524 and 578 mumol/kg for AHH and EROD, respectively, and TCDD was greater than 1.5 X 10(5) more potent than MCDF as an agonist for this response. Cotreatment of the female rats with MCDF (20, 50, or 100 mumol/kg) and TCDD (6.4 nmol/kg) showed that MCDF partially antagonized the induction of AHH and EROD by TCDD and this corresponded with results previously reported in the male rat. Like TCDD, MCDF also caused a dose-response decrease in uterine and hepatic cytosolic and nuclear estrogen (ERc and ERn) and progesterone (PRc and PRn) receptor levels. The relative TCDD/MCDF potencies for the reduction of uterine ERc, ERn, PRc, and PRn levels were 293, 569, 560, and 459, respectively, and comparable potency ratios (693, 409, 405, and 424, respectively) were observed in the liver. Since MCDF was active as an antiestrogen at dose levels which caused only minimal induction of hepatic monooxygenases, it is unlikely that induction of these enzymes and the subsequent increased metabolism of estradiol play a role in the antiestrogenic effects of MCDF (or TCDD). The reasons for the differences in the relative potency of MCDF for the "traditional" Ah receptor-mediated response (i.e., AHH induction) and the modulation of steroid hormone receptor binding levels are unknown. MCDF, a compound which exhibits relatively high TCDD receptor binding activity and low toxicity, represents a new class of nontoxic halogenated aromatic antiestrogens that can be utilized to further probe the mechanism of this response in model systems.

Animals↗

PubMed1988

Immunosuppressive activities of polychlorinated dibenzofuran congeners: quantitative structure-activity relationships and interactive effects.

The dose-response immunosuppressive effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), 2,3,4,7,8- and 1,2,3,7,9-pentachlorodibenzofuran (PeCDF), 2,3,7,8- and 1,3,6,8-tetrachlorodibenzofuran (TCDF) on the splenic plaque-forming cell (PFC) response to sheep red blood cells were determined in C57BL/6 mice. The ED50 values for immunosuppression were 2.4, 3.0, 14.0, 710, and 35,700 nmol/kg for 2,3,7,8-TCDD, 2,3,4,7,8-PeCDF, 2,3,7,8-TCDF, 1,2,3,7,9-PeCDF, and 1,3,6,8-TCDF, respectively, and the results confirmed that lateral chlorine substitutions were important structural determinants for the toxicity of the polychlorinated dibenzofuran congeners. Interaction of both 2,3,7,8-TCDD and 2,3,4,7,8-PeCDF with subimmunotoxic doses of 1,3,6,8-TCDF resulted in significant antagonism of the immunotoxic effects of both 2,3,7,8-TCDD and 2,3,4,7,8-PeCDF. Previous studies have also demonstrated that 1,3,6,8-TCDF also antagonizes the induction of aryl hydrocarbon hydroxylase by 2,3,7,8-TCDD and analysis of competitive receptor binding studies suggests that 1,3,6,8-TCDF acts as a competitive partial antagonist of the action of 2,3,7,8-TCDD. The antagonism of 2,3,7,8-TCDD immunosuppression was found to be dependent on the timing of administration of 1,3,6,8-TCDF. Using a protocol in which 2,3,7,8-TCDD is administered 5 days prior to the antigen and 9 days prior to assessing the splenic PFC response, it was possible to partially antagonize the immunosuppressive effects of 2,3,7,8-TCDD by administering the antagonist up to 5 days after the initial dose of the toxin. Administration of 1,3,6,8-TCDF after the antigen does not afford any significant protection from the effects of 2,3,7,8-TCDD and these results are consistent with the hypothesis that 2,3,7,8-TCDD modulates some early event in B-cell differentiation. However, these results do not exclude a role for 2,3,7,8-TCDD in modulating other cellular processes associated with the PFC response.

Animals↗

PubMed1988

Biologic and toxic effects of polychlorinated dibenzo-p-dioxin and dibenzofuran congeners in the guinea pig. Quantitative structure-activity relationships.

The dose-response effects of 2,3,7,8-tetrachorodibenzo-p-dioxin, 1,3,7,8-tetrachlorodibenzo-p-dioxin, 1,2,4,7,8-pentachlorodibenzo-p-dioxin, 2,3,4,7,8-, 1,2,3,7,9-, and 2,3,4,7,9-pentachlorodibenzofuran on body weight loss and hepatic microsomal aryl hydrocarbon hydroxylase (AHH) and ethoxyresorufin O-deethylase (EROD) induction were determined in the immature male guinea pig. The ED50 values for each compound were measured for the three in vivo responses. The quantitative structure-activity relationships clearly illustrated that the most toxic congeners were substituted in the lateral 2, 3, 7 and 8 positions, and removal of a lateral chlorine group substantially reduced the potency of the resulting compound. The most toxic congener in this series was 2,3,7,8-tetrachlorodibenzo-p-dioxin in which the in vivo ED50 values for AHH and EROD induction and body weight loss were 2.8 X 10(-10), 9.3 X 10(-11) and 5.6 X 10(-9) mol/kg. The structure-activity relationships observed in this study were comparable to those previously reported in rats and rat hepatoma H-4-II E cells in culture. Moreover, there was an excellent linear correlation between in vivo -log ED50 values for body weight loss, AHH and EROD induction and the corresponding in vitro -log EC50 data for AHH induction in rat hepatoma cells [S. Safe, Chemosphere 16, 791 (1987)].

Animals↗

PubMed1988

Comparative activities of 2,3,7,8-tetrachlorodibenzo-p-dioxin and progesterone as antiestrogens in the female rat uterus.

The comparative antiestrogenic effects of progesterone and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on cytosolic and nuclear estrogen (ERc and ERn, respectively) and progesterone (PRc and PRn, respectively) receptor levels were determined in female Long Evans rats. Estradiol treatment typically increases ER and PR levels in target cells or tissues and these proteins have been proposed as markers of estrogen action. 2,3,7,8-TCDD causes a dose-dependent decrease in uterine ERc, ERn, PRc, and PRn levels which persist up to 7 days. Progesterone treatment caused significant decreases in uterine ERc, ERn, and PRn levels; however, after 7 days, the effects of the hormone on receptor levels were diminished. The effects of 2,3,7,8-TCDD and progesterone on hepatic ER and PR levels were comparable to those observed in the uterus. Treatment of the rats with estradiol (5 micrograms/kg), estradiol (5 micrograms/kg) plus progesterone (1 mg/animal), or 2,3,7,8-TCDD (80 micrograms/kg) showed that both progesterone and 2,3,7,8-TCDD significantly antagonized the estradiol-mediated increases in uterine (and hepatic) ERc, ERn, PRc, and PRn levels and for 2,3,7,8-TCDD the decreased receptor levels persisted for up to 7 days. In vitro studies with freshly isolated uterine strips demonstrated that both 2,3,7,8-TCDD and progesterone antagonized the estradiol-mediated increases in ER and PR levels. Previous studies suggest that the antiestrogenic activity of progesterone is due, in part, to the induction of proteins which are involved in decreasing ERn levels in target cells. Moreover in the uterine strip assay procedure, it was previously shown and reproduced in this study that the decrease in uterine ERn by progesterone was inhibited by both protein and RNA synthesis inhibitors over a 4-hr incubation period. In contrast, the 2,3,7,8-TCDD-mediated decrease in uterine ERn was inhibited only by actinomycin D and not by cycloheximide or puromycin. These in vitro studies thus confirm that both progesterone and 2,3,7,8-TCDD exhibit comparable antiestrogenic effects in vivo and in vitro; however, the results suggest that these effects are expressed through different mechanisms.

Animals↗

PubMed1988

Applications of the in vitro aryl hydrocarbon hydroxylase induction assay for determining "2,3,7,8-tetrachlorodibenzo-p-dioxin equivalents": pyrolyzed brominated flame retardants.

The pyrolysis of brominated flame retardants FR 300 BA (decabromobiphenyl) ether, FireMaster BP-6 (polybrominated biphenyls), Bromkal 70-5-DE (primarily pentabromodiphenylether), Bromkal 70-DE (primarily penta and tetrabromodiphenylether) and Bromkal G1 (pentabromodiphenylether) resulted in the formation of relatively high levels of polybrominated dibenzofurans (PBDFs) and dibenzo-p-dioxins (PBDDs as determined by gas chromatography-mass spectrometric analysis. The dose response EC50 values for the induction of aryl hydrocarbon hydroxylase (AHH) and ethoxyresorufin O-deethylase (EROD) by the flame retardant pyrolysates was determined in rat hepatoma H-4-II E cells and compared to the relative induction activities of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and the concentrations of "2,3,7,8-TCDD equivalents" were calculated. The range of "2,3,7,8-TCDD equivalents" levels (micrograms/g or ppm) derived from values obtained from the AHH and EROD bioassays for each of the pyrolyzed flame retardant samples was: 174-194, 480-1400, 2140-4680, 6740-8780 and 3920-5260 ppm for FR 300 BA, FireMaster BP-6, Bromkal 70 DE, Bromkal 70-5 DE and Bromkal G1, respectively. The in vivo dose-response effects of 2 pyrolyzed flame retardants were determined in immature male Wistar rats and compared to the dose-response activities of 2,3,7,8-TCDD. The in vivo responses which were measured included hepatic microsomal AHH and EROD induction, body weight loss and thymic atrophy. For the pyrolyzed FireMaster BP-6 and Bromkal 70-5 DE samples, the range of calculated in vivo "2,3,7,8-TCDD equivalents" (ppm in sample) for the 4 in vivo bioassays was 520-1780 ppm and 3860-8960 ppm, respectively. The excellent overlap between the in vivo and in vitro 2,3,7,8-TCDD equivalents for the 2 flame retardant pyrolysate extracts supports the utility of the in vitro induction bioassay for quantitatively determining "2,3,7,8-TCDD equivalents" for mixtures containing toxic halogenated aryl hydrocarbons.

Animals↗