L. Safe

High-resolution PCB analysis: synthesis and chromatographic properties of all 209 PCB congeners

The synthesis and spectroscopic properties of all the mono-, di-, tri-, tetra-, penta-, hexa-, and heptachlorobipheynls are reported and the synthesis of all 209 polychlorinated biphenyls (PCBs) is completed. The retention times and molar response factors of the 209 PCBs were determined relative to a reference standard, octachloronaphthalene. The retention times for these compounds generally increased with increasing chlorine content, and it was apparent that within a series of isomers there was an increase in retention time with increasing meta and para and decreasing ortho substitution. By use of a 50-m narrow bore fused silica capillary column coated with SE-54, it was possible to separate 187 PCB congeners, and only 11 pairs of compounds were not fully resolved. With some additional analytical improvements, isomer-specific PCB analysis can be utilized to determine the composition of commercial PCBs and accurately follow the fate and distribution of these pollutants within the global ecosystem.

PCBs: Structure-Function Relationships and Mechanism of Action

Numerous reports have illustrated the versatility of polychlorinated biphenyls (PCBs) and related halogenated aromatics as inducers of drug-metabolizing enzymes and the activity of individual compounds are remarkably dependent on structure. The most active PCB congeners, 3,4,4′,5-tetra-, 3,3′,4,4′-tetra-, 3,3′,4,4′,5-penta- and 3,3′,4,4′,5,5′-hexachlorobiphenyl, are substituted at both para and at two or more meta positions. The four coplanar PCBs resembled 3-methylcholanthrene (3-MC) and 2,3,7,8-tetrachlorodibenzo-p-dioxin (2,3,7,8-TCDD) in their mode of induction of the hepatic drug-metabolizing enzymes. These compounds induced rat hepatic microsomal benzo(a)pyrene hydroxylase (aryl hydrocarbon hydroxylase, AHH) and cytochromes P-450a, P-450c and P-450d. 3,4,4′,5-Tetrachlorobiphenyl, the least active coplanar PCB, also induced dimethylaminoantipyrine N-demethylase and cytochromes P-450b+e and resembled Aroclor 1254 as an inducer of the mixed-function oxidase system. Like Aroclor 1254, all the mono-ortho- and at least eight di-ortho-chloro analogs of the coplanar PCBs exhibited a “mixed-type” induction pattern and induced microsomal AHH, dimethylaminoantipyrine NM-demethylase and cytochromes P-450a–P-450e. Quantative structure–activity relationships (QSARs) within this series of PCBs were determined by comparing their AHH induction potencies (EC50) in rat hepatoma H-4-II-E cells and their binding affinities (ED50) for the 2,3,7,8-TCDD cytosolic receptor protein. The results showed that there was an excellent correlation between AHH induction potencies and receptor binding avidities of these compounds and the order of activity was coplanar PCBs (3,3′,4,4′-tetra-, 3,3′,4,4′,5-penta- and 3,3′,4,4′,5,5′-hexachlorobiphenyls) > 3,4,4′,5-tetrachlorobiphenyl ~ mono-ortho coplanar PCBs > di-ortho coplanar PCBs. It was also apparent that the relative toxicities of this group of PCBs paralleled their biological potencies. The coplanar and mono-ortho coplanar PCBs also exhibit differential effects in the inbred C57BL/6J and DBA/2J mice. These compounds induce AHH and cause thymic atrophy in the former “responsive” mice whereas at comparable or higher doses none of these effects are observed in the nonresponsive DBD/2J mice. Since the responsiveness of these two mice strains is due to the presence of the Ah receptor protein in the C57BL/6J mice and its relatively low concentration in the DBA/2J mice, the results for the PCB cogeners support the proposed receptor-mediated mechanism of action. Although the precise structural requirements for ligand binding to the receptor have not been delineated, the halogenated aromatic hydrocarbons which exhibit the highest binding affinities for the receptor protein are approximate isostereomers of 2,3,7,8-TCDD. 2,3,4,4′,5-Pentachlorobiphenyl elicits effects which are qualitatively similar to that of TCDD and the presence of the lateral 4′-substituent is required for this activity. Thus the 4′-substituted 2,3,4,5-tetrachlorobiphenyls have been used as probes for determining the substituent characteristics which favor binding to the receptor protein. Multiple regression analysis of the competitive binding EC50 values for 13 substituents gave the following equation: log (1/EC50) = 1.53σ + 1.47π + 1.09 HB + 4.08 where σ is electronegativity, π is hydrophobicity, HB is hydrogen bonding and r is the correlation coefficient (r = 0.978). The utility of this equation in describing ligand:receptor interactions and correlations with toxicity are being studied with other halogenated hydrocarbons and PAHs.

Polychlorinated dibenzofurans (PCDFs): Effects of structure on binding to the 2,3,7,8-TCDD cytosolic receptor protein, AHH induction and toxicity

The effects of structure on the activity of 26 polychlorinated dibenzofurans (PCDFs) as competitive ligands for the 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) rat hepatic cytosolic receptor protein were determined in a dose-response fashion. The ED50 values for these compounds varied 100 000-fold and the most active PCDFs were substituted in the 2,3,7 and 8 lateral positions; the ED50 for the most active PCDF, 2,3,4,7,8-pentachlorodibenzofuran was 1.5 X 10(-8) M which was only slightly less active than 2,3,7,8-TCDD (1.0 X 10(-8) M). A comparison of the binding affinities of several isomer pairs also indicated the relative importance of chlorine substitution at C-4 (or C-6) compared to C-1 (or C-9). Moreover, for some isomers it is apparent that C-4 (or C-6) substituents are more active than lateral substituents for facilitating ligand binding to the receptor protein. This is illustrated by the relative binding potencies of the following isomer pairs: 1,2,4,6,7-/1,2,4,7,8 = 19.2; 2,6,7-/2,3,8- = 2.2; 1,3,6-/1,3,8- = 19. Most of the PCDF structure-activity effects noted above were also observed for the induction of aryl hydrocarbon hydroxylase (AHH) and ethoxyresorufin O-deethylase (EROD) in rat hepatoma H-4-II-E cells in culture. The most active compounds were also substituted in the lateral 2,3,7 and 8 positions and a comparison of C-4 (or C-6) vs. C-1 (or C-9) substituted PCDFs confirmed the higher induction potencies for most of the former group of compounds. The in vitro quantitative structure-activity data were complemented by in vivo studies which determined the relative activities of selected PCDFs as inducers of hepatic microsomal cytochrome P-448 dependent monooxygenases and their effects on body weight gain and thymus weights in immature male Wistar rats. The results indicated that for 2 series of isomers, namely the 2,3,4,7,8-, 1,2,4,7,8- and 1,2,4,7,9-pentachlorodibenzofurans and the 2,3,7,8-, 2,3,4,8- and 1,2,4,8-tetrachlorodibenzofurans, their biologic and toxic potencies were dependent on one major structural factor, the number of lateral chloro substituents. These results support the proposed role of the cytosolic receptor protein in mediating the biologic and toxic effects of the PCDFs.

Polychlorinated dibenzo-p-dioxins: quantitative in vitro and in vivo structure-activity relationships.

There were marked effects of structure on the activities of 14 polychlorinated dibenzo-p-dioxins (PCDDs) as competitive ligands for the 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) receptor and as inducers of aryl hydrocarbon hydroxylase (AHH) and ethoxyresorufin O-deethylase (EROD) in rat hepatoma H-4-II E cells in culture. 2,3,7,8-TCDD was the most active compound in both assays and several PCDD congeners which were fully substituted in the lateral 2, 3, 7 and 8 positions but also contained additional chlorosubstituents in non-lateral 1, 4, 6 and 9 positions were less active. It was also evident that there was a decrease in in vitro binding and induction activities with decreasing lateral chlorine substitution. Although comparable structure-activity relationships (SARs) for the PCDDs were observed for the induction and receptor binding assays, there was not a linear or rank order correlation between the 2 sets of data. Several in vivo biologic and toxic activities of 2,3,7-trichloro-, 2,3,7,8- and 1,3,7,8-tetrachloro-, 1,2,4,7,8- and 1,2,3,7,8-pentachloro- and 1,2,3,4,7,8-hexachlorodibenzo-p-dioxin were determined in a dose-response fashion in immature male Wistar rats. The ED50 values for hepatic microsomal AHH and EROD induction, body weight loss and thymic atrophy were obtained. There was an excellent linear correlation between the -log EC50 values for AHH or EROD induction in cell culture and the -log ED50 values for enzyme induction, body weight loss and thymic atrophy in the rat. The in vitro enzyme induction data could be used to quantitatively estimate the toxicity of the PCDD congeners in the rat: this latter correlation has previously been observed for a series of polychlorinated dibenzofurans.

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.

Polybrominated dibenzo-p-dioxins and related compounds: Quantitative in vivo and in vitro structure-activity relationships

The effects of structure on the in vitro receptor binding affinities, aryl hydrocarbon hydroxylase (AHH) and ethoxyresorufin O-deethylase (EROD) induction potencies in rat hepatoma cells were determined for the following compounds: 2-bromo-, 2,7/2,8-dibromo-, 2,3,7-tribromo-, 2,4,6,8/1,3,7,9-tetrabromo-, 2,3,7,8-tetrabromo-, 1,3,7,8-tetrabromo-, 1,2,3,7,8-pentabromo-, 1,2,4,7,8-pentabromo-, 2,3-dibromo-7,8-dichloro-, 2,8-dibromo-3,7-dichloro- and 2-bromo-3,7,8-trichlorodibenzo-p-dioxin. The structure-activity relationships (SARs) for the polybrominated dibenzo-p-dioxins (PBDDs) were comparable for both in vitro responses: the most active compounds were substituted only in the lateral 2,3,7 and 8 position and the addition of non-lateral or removal of lateral halogen substituents reduced the activity of the resultant compound. The biologic and toxic effects of 2,3,7,8-tetrabromo-, 1,3,7,8-tetrabromo-, 1,2,4,7,8-pentabromo-1,2,3,7,8-pentabromo-, 2-bromo-3,7,8-trichloro- and 2,3-dibromo-7,8-dichlorodibenzo-p-dioxin on several receptor-mediated responses (thymic atrophy, body weight loss, hepatic microsomal AHH and EROD induction) were determined in a dose-response fashion in immature male Wistar rats. A comparison of the ED50 values for the in vivo responses demonstrated that the SARs for the PBDDs and brominated polychlorinated dibenzo-p-dioxins were comparable to those observed for in vitro receptor binding and AHH induction. Moreover, there was an excellent linear correlation between the -log EC50 (in vitro AHH induction) vs. the in vivo -log ED50 (thymic atrophy) and -log ED50 (body wt loss) correlation coefficient, r = 0.97 for all 2 correlations).

Polychlorinated Biphenyls: Environmental Occurrence and Analysis

Polychlorinated biphenyls (PCBs) are complex mixtures which have been identified in every component of the global ecosystem. This chapter discusses the distribution of PCBs in the environment and points out recent analytical advances which now permit high resolution congener-specific analysis of PCBs in diverse analytes using high resolution capillary gas chromatographic techniques. The biologic and toxic effects of PCBs are structure-dependent and the adverse environmental and human health impacts of the different mixtures of PCBs are related to the individual components of these mixtures and their interactions. The chapter points out that high resolution PCB analysis will now permit the unequivocal identification and quantitation of the individual PCBs in environmental samples and this data can be used for more accurate risk assessment

Synthesis and aryl hydrocarbon receptor binding properties of radiolabeled polychlorinated dibenzofuran congeners

Microchlorination of 1,4,9[3H]dibenzofuran gave several polychlorinated dibenzofuran (PCDF) products and 2,3,7,8-[3H]tetrachlorodibenzofuran (TCDF), 1,2,3,7,8-[3H]pentachlorodibenzofuran (PeCDF), and 1,2,3,6,7,8-/1,2,3,4,7,8-hexachlorodibenzofuran (HCDF) of high specific activity (57, 34, and 32.5 Ci/mmol, respectively) were purified by preparative high-pressure liquid chromatography. These compounds were investigated as radioligands for the rat liver cytosolic aryl hydrocarbon (Ah) receptor protein. Like 2,3,7,8-[3H]tetrachlorodibenzo-p-dioxin (TCDD), the radiolabeled PCDF congeners exhibited saturable binding with the receptor protein and sucrose density gradient analysis of the radiolabeled ligand-receptor complexes gave specific binding peaks with comparable sedimentation profiles. The rank order of radioligand binding affinities (Kd values) was 2,3,7,8-TCDD greater than 2,3,7,8-TCDF greater than 1,2,3,6,7,8-HCDF greater than 1,2,3,7,8-PeCDF and the maximum difference in Kd values for the four radioligands was less than 13-fold (0.44-5.9 nM). The interactions of the PCDF radioligands with the cytosolic receptor all exhibited saturable binding curves and linear Scatchard plots and the slopes of their Hill plots were in the range 1.0-1.1, thus indicating that cooperativity was not a factor in these binding interactions. The relative stabilities and dissociation kinetics of the radioligand-receptor complexes were highly dependent on the structure of the radioligand. The dissociation curves of the 2,3,7,8-[3H]TCDD and PCDF receptor complexes were biphasic and this suggests that there may be a temporal shift in ligand binding affinities. However, the rates of dissociation did not correlate with the rank order of ligand binding affinities. The stabilities of the radioligand-receptor complexes were also dependent on the structures of the radioligands; for example, the 2,3,7,8-[3H]TCDD-receptor complex degraded more rapidly than the PCDF-receptor complex and these relative stabilities were clearly not related to the Kd values or the relative in vivo or in vitro biologic potencies of these halogenated aryl hydrocarbons.

Mechanism of action of 2,3,7,8-tetrachlorodibenzo-p-dioxin antagonists: Characterization of 6-[125I]methyl-8-iodo-1, 3-dichlorodibenzofuran-Ah receptor complexes☆

6-Methyl-8-iodo-1,3,-dichlorodibenzofuran (I-MCDF) and its radiolabeled analog [125I]MCDF have been synthesized and used to investigate the mechanism of action of 1,3,6,8-substituted dibenzofurans as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) antagonists. Like 6-methyl-1,3,8-trichlorodibenzofuran (MCDF), I-MCDF partially antagonized the induction by TCDD of microsomal aryl hydrocarbon hydroxylase (AHH) and ethoxyresorufin O-deethylase (EROD) activities in rat hepatoma H-4-II E cells and male Long-Evans rat liver. Incubation of rat liver cytosol with [125I]MCDF followed by velocity sedimentation analysis on sucrose gradients gave a specifically bound peak which sedimented at 9.6 S. This radioactive peak was displaced by coincubation with a 200-fold excess of unlabeled I-MCDF, 6-methyl-1,3,8-trichlorodibenzofuran (MCDF), 2,3,7,8-tetrachlorodibenzofuran (TCDF), and benzo [a]pyrene. Based on the velocity sedimentation results and the elution profile from a Sephacryl S-300 gel permeation column, the Stokes radius and apparent molecular weights of the cytosolic [125I]MCDF-Ah receptor complex were 6.5 nm and 259,200, respectively. In addition, the nuclear [125I]MCDF-receptor complex eluted at a salt concentration of 0.29 M KCl from a DNA-Sepharose column. Velocity sediment analysis of the nuclear [125I]MCDF-Ah receptor complex from rat hepatoma H-4-II E cells gave a specifically bound peak at 5.6 +/- 0.8 S. All of these properties were similar to those observed using [3H]TCDD as the radioligand. In addition, there were several ligand-dependent differences observed in the properties of the I-MCDF and TCDD receptor complexes; for example, the [125I]MCDF rat cytosolic receptor complex was unstable in high salt buffer and was poorly transformed into a form with increased binding affinity on DNA-Sepharose columns; Scatchard plot analysis of the saturation binding of [3H]TCDD and [125I]MCDF with rat hepatic cytosol gave KD values of 1.07 and 0.13 nM and Bmax values of 137 and 2.05 fmol/mg protein, respectively. The nuclear extract from rat hepatoma H-4-II E cells treated with I-MCDF or TCDD interacted with a dioxin-responsive element in a gel retardation assay. These results suggest that the mechanism of antagonism may be associated with competition of the antagonist receptor complex for nuclear binding sites.

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.