D. Davis

Aroclor 1254 as a 2,3,7,8-tetrachlorodibenzo-p-dioxin antagonist: Effects on enzyme induction and immunotoxicity

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) and Aroclor 1254 induced the cytochrome P-450 dependent monooxygenases, aryl hydrocarbon hydroxylase (AHH) and ethoxyresorufin O-deethylase (EROD) in rat hepatoma H-4-II E cells and C57BL/6J mice. It has been proposed that both Aroclor 1254 and 2,3,7,8-TCDD induce these enzymes via a common mechanism which features initial binding to the aryl hydrocarbon (Ah) cytosolic receptor protein. The major difference between these compounds was the relative potency (i.e. 2,3,7,8-TCDD much greater than Aroclor 1254). Cotreatment of rat hepatoma H-4-II E cells or C57BL/6J mice with a dose of 2,3,7,8-TCDD which submaximally induces AHH and EROD and a dose of Aroclor 1254 which exhibited little or no induction activity resulted in significant antagonism of the induction effects of 2,3,7,8-TCDD. For example, cotreatment of C57BL/6J mice with 2,3,7,8-TCDD (15 nmol/kg) and Aroclor 1254 (25, 75 and 150 mumol/kg) resulted in up to 23% antagonism of AHH induction by 2,3,7,8-TCDD. Moreover, cotreatment with a higher dose of the 2,3,7,8-TCDD agonist (30 or 50 nmol/kg) partially reversed some of the antagonism by Aroclor 1254. In vivo antagonism was observed only at Aroclor 1254/2,3,7,8-TCDD molar ratios of 1667:1, 5000:1 and 10,000:1. Administration of 2,3,7,8-TCDD (3.72 nmol/kg) to C57BL/6J mice resulted in a 76% decrease in the splenic plaque forming cell response to sheep red blood cells. This T-cell mediated immunotoxic effect of 2,3,7,8-TCDD segregates with the Ah locus. In contrast, administration of 5, 15, 75 and 150 mumol/kg of Aroclor 1254 resulted in impairment of the immune response only at the highest dose level. However, cotreatment of mice with 2,3,7,8-TCDD (3.72 nmol/kg) and Aroclor 1254 (5, 15 or 75 mumol/kg) resulted in no significant decrease in the plaque forming cell response and complete protection from the immunotoxicity of 2,3,7,8-TCDD. Cotreatment of the mice with Aroclor 1254 (75 mumol/kg) and a higher dose of the 2,3,7,8-TCDD agonist resulted in partial reversal of the protective effects of Aroclor 1254. The in vitro and in vivo data suggest that within specific antagonist/agonist dose ratios, Aroclor 1254 can antagonize at least 2 Ah receptor-mediated effects of 2,3,7,8-TCDD, namely AHH induction and immunotoxicity.

2,2′,4,4′,5,5′-Hexachlorobiphenyl as a 2,3,7,8-tetrachlorodibenzo-p-dioxin antagonist in C57BL6J mice

Abstract At doses as high as 750 to 1000 μmol/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-[ 3 H ] 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 (I 2 -TCBP) (400 or 1000 μmol/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 I 2 -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 [ 3 H ] 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′-[ 125 I 2 ] diiodo -2,2′,5,5′- tetrachlorobiphenyl ([ 125 I 2 ] 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 125 I 2 - 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.

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.

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.

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.

Development and validation of in vitro bioassays for 2,3,7,8-TCDD equivalents

Abstract Toxic halogenated aryl hydrocarbons are industrial compounds and/or by-products which have been detected as complex mixtures in almost every component of the global ecosystem. Included in this class of chemical pollutants are the halogenated (Cl/Br) biphenyls (PCBs/PBBs), dibenzo-p-dioxins (PCDDs/PBDDs) and dibenzofurans (PCDFs/PBDFs). Rat hepatoma H-4-II E cells are highly sensitive to the induction of aryl hydrocarbon hydroxylase (AHH) and ethoxyresorufin O-deethylase (EROD) by 2,3,7,8-TCDD and related toxic halogenated aromatics and for > 25 of these compounds there was an excellent correlation (r = 0.85 to 0.93) between their in vitro pEC 50 values for induction and their pED 50 values for in vivo effects in the rat (AHH induction, body weight loss and thymic atrophy). Moreover, for selected PCB, PCDD and PCDF congeners comparable linear correlations between in vitro induction activity and in vivo toxicities in the guinea pig (AHH induction and body weight loss) and mouse (immunotoxicity) were also observed. The data confirm the utility of the in vitro induction bioassay for toxic halogenated aromatics. The results of these studies permit the calculation of estimated “2,3,7,8-TCDD equivalent ranges” for most of the important toxic halogenated aromatics and provide a more rational basis for risk assessment of these compounds and their mixtures.

The Structure-Dependent Effects of Heptachlorodibenzofuran Isomers in Male C57BL/6 Mice: Immunotoxicity and onooxygenase Enzyme Induction

The dose-response effects of the 1,2,3,4,6,7,8-, 1,2,3,4,7,8,9-, 1,2,3,4,6,8,9-, and 1,2,3,4,6,7,9-heptachlorodibenzofurans (HpCDFs) on the splenic plaque-forming cell (PFC) response to sheep erythrocytes and on the induction of hepatic microsomal aryl hydrocarbon hydroxylase (AHH) and ethoxyresorufin-O-deethylase (EROD) activities were determined in male C57BL/6 mice. The ED50 values for the decrease in the PFCs/spleen, the number of PFCs/10(6) viable cells, and the induction of AHH and EROD activities were 1,2,3,4,6,7,8-HpCDF, 0.011, 0.018, 0.11, and 0.315 mumol/kg, respectively; 1,2,3,4,7,8,9-HpCDF, 0.012, 0.054, 0.70, and 0.20 mumol/kg, respectively; 1,2,3,4,6,7,9-HpCDF, 1.2, 1.3, greater than 43, and greater than 43 mumol/kg, respectively; 1,2,3,4,6,8,9-HpCDF, 1.5, 3.4, 22, and 22 mumol/kg, respectively. It was apparent from these studies that the 2,3,7,8-substituted HpCDF isomers (1,2,3,4,6,7,8- and 1,2,3,4,7,8,9-) were significantly more potent than the compounds which contained only three lateral C1 groups. These results were obtained using a multiple dosing regimen in which 10 separate doses of the HpCDF isomers were administered to the mice by intraperitoneal injection over a period of 12 days. However, when the mice were treated with a single dose of an HpCDF congener, which was equivalent to the total dose used in the multiple dose study, the responses were comparable. A comparison of the relative immunotoxic potencies of the 2,3,7,8-substituted HpCDFs and 2,3,7,8-tetrachlorodibenzo-p-dioxin showed that the latter compound was approximately 10 times more active than the HpCDFs.

Interactions of 2,3,7,8-TCDD and PCB mixtures/congeners: Immunotoxicity studies

Abstract Several commercial PCBs, including Aroclors 1260, 1254, 1248, 1242 and 1232, were immunotoxic in C57BL/6 mice, however the results showed that the lower chlorinated biphenyls (Aroclor 1232 and 1242) were less toxic than the higher chlorinated mixtures. Treatment of C57BL/6J mice with an immunotoxic dose of 2,3,7,8-TCDD (3.7 nmol/kg) and a dose (25 mg/kg) of the commercial PCBs which was not immunotoxic showed that the PCB mixtures antagonized the 2,3,7,8-TCDD-mediated decrease in the plaque-forming cell response to sheep erythrocytes. The structure-dependent effects of individual PCB congeners to act as 2,3,7,8-TCDD antagonists were also investigated and only 2 compounds, namely 2,2′,4,4′,5,5′-and 2,3,3′,4,5,5′-hexachlorobiphenyl exhibited antagonist activity comparable to that observed for Aroclor 1254 as a 2,3,7,8-TCDD antagonist. The implications of these results will be discussed.

Aroclor 1254 as a 2,3,7,8-tetrachlorodibenzo-p-dioxin antagonist in mice

Abstract Both Aroclor 1254, a commercial polychlorinated biphenyl (PCB), and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) elicit common aryl hydrocarbon (Ah) receptor-mediated effects including the induction of aryl hydrocarbon hydroxylase (AHH), a cytochrome P-450-dependent monooxygenase, the inhibition of the T-cell dependent plaque forming cells in response to sheep red blood cells (in C57BL/6 mice) and cleft palate in C57BL/6 mice. However based on ED50 values from dose-response studies with Aroclor 1254 and 2,3,7,8-TCDD, it was apparent that the latter compound is at least 105 times more potent. Cotreatment of rat hepatoma H-4-II E cells or C57BL/6 mice with an ED80–100 dose of 2,3,7,8-TCDD plus various sub-toxic (or effective) doses of Aroclor 1254 clearly shows that the commercial PCB can significantly antagonize 2,3,7,8-TCDD-mediated AHH induction (in vivo and in vitro), immunotoxicity and teratogenicity. In vitro Ah receptor binding studies suggests that Aroclor 1254 competitively inhibits the Ah receptor binding of 2,3,7,8-TCDD and this may account for the antagonist activity of the commercial PCB.

6-methyl-1,3,8-trichlorodibenzofuran (MCDF) as a 2,3,7,8-TCDD antagonist in C57BL/6 mice

MCDF exhibits moderate affinity for the Ah receptor in both mice and rats; however, it is only a weak Ah receptor agonist in both species. In contrast, to previous studies in rats, MCDF was a relatively poor antagonist of TCDD-induced monooxygenase activities in mice, and over a dose range of 10–500 μmol/kg, significant inhibition was observed only at a dose of 50 μol/kg. These dose-response interactive studies in C57BL/6 mice showed that MCDF significantly antagonized the TCDD-mediated induction of hepatic microsomal aryl hydrocarbon hydroxylase (AHH), 14% inhibition, and ethoxyresorufin-O-deethylase (EROD), 17% inhibition. MCDF was also a weak agonist for teratogenicity (cleft palate) and immunotoxicity (inhibition of the plaque-forming cell response to sheep erythrocytes) in C57BL/6 mice. Cotreatment of mice with an effective dose of 2,3,7,8-TCDD (ED70–100) and a subeffective dose of MCDF demonstrated that MCDF significantly antagonized 2,3,7,8-TCDD-induced teratogenicity and immunotoxicity in the mice.