B. Keys

The cytosolic receptor binding affinities and ahh induction potencies of 29 polynuclear aromatic hydrocarbons

The dose-response rat hepatic cytosolic receptor-binding avidities, aryl hydrocarbon hydroxylase (AHH) and ethoxyresorufin O-deethylase (EROD) induction potencies in rat hepatoma H-4-II E cells in culture were determined for 29 polynuclear aromatic hydrocarbons. It was apparent that the magnitude of the EC50 values for these in vitro responses were strongly dependent on structure. Dibenz[a,h]anthracene (1.6 X 10(-8) M), 7-methylbenz[a]anthracene (1.6 X 10(-8) M), 3-methylcholanthrene (2.8 X 10(-8) M) and picene (4.5 X 10(-8) m) exhibited the highest affinity for the receptor protein and these compounds were only 5-fold less active the 2,3,7,8-tetrachlorodibenzo-p-dioxin (1 X 10(-8) M). All of the compounds which were active in the receptor-binding and monooxygenase enzyme-induction assays possessed one common structural feature, namely the presence of a phenanthrene structure fused with at least 1 benzo ring. The results also demonstrated that there was not any apparent correlation between the receptor-binding avidities and in vitro monooxygenase enzyme-induction potencies for the most active polynuclear aromatic hydrocarbons.

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 dibenzofurans (PCDFs): Correlation between in vivo and in vitro structure-activity relationships

Fifteen polychlorinated dibenzofuran (PCDF) congeners were administered in a dose-response fashion to immature male Wistar rats and ED50 values for body weight loss, thymic atrophy and the induction of the hepatic microsomal cytochrome P-448-dependent monooxygenases, aryl hydrocarbon hydroxylase (AHH) and 4-chlorobiphenyl hydroxylase were determined. There was an excellent correlation between the in vivo quantitative structure-activity relationships for these PCDFs and their in vitro activities as AHH inducers in rat hepatoma H-4-II E cells and as ligands for the 2,3,7,8-TCDD receptor protein. A comparison of isomers which differ at all 4 positions in the dibenzofuran ring system indicated that chlorine substitution at each position contributed differentially to the overall molecular activity [C-3 (or C-7) greater than C-2 (or C-8) greater than C-4 (or C-6) greater than C-1 (or C-9)]. There was also an excellent linear correlation between a plot of the -log ED50 for body weight loss vs. -log EC50 for in vitro AHH induction (correlation coefficient, r = 0.96) and -log ED50 for thymic atrophy vs. -log EC50 for in vitro AHH induction (correlation coefficient, r = 0.88). Since body weight loss and thymic atrophy in the rat are representative toxic responses to PCDFs and related toxic halogenated aryl hydrocarbons, the correlations noted above support the use of the in vitro AHH induction assay as a short term quantitative test system for this class of toxic halogenated aryl hydrocarbons.

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.

Polychlorinated dibenzofurans as 2,3,7,8-tcdd antagonists: In vitro inhibition of monooxygenase enzyme induction

2,4,6,8- and 1,3,6,8-tetrachlorodibenzofuran (TCDF) competitively displace [3H]2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) from the rat cytosolic receptor protein and their EC50 values were 1.5 X 10(-6) and 1.25 X 10(-7) M, respectively. In contrast to their relatively high binding avidities these TCDF isomers were poor inducers of benzo[a]pyrene hydroxylase and ethoxyresorufin O-deethylase in rat hepatoma H-4-II E cells in culture (EC50 greater than 10(-5) M). Coadministration of different concentrations of 2,4,6,8- and 1,3,6,8-TCDF (10(-5), 10(-6) and 10(-7) M) with 2 X 10(-10) M, 2,3,7,8-TCDD (a dose which elicits 80% of the maximal induction response) resulted in significant decreases in the expected (additive) induction of benzo[a]pyrene hydroxylase and ethoxyresorufin O-deethylase by the mixture. Thus the partial agonists, 1,3,6,8- and 2,4,6,8-TCDF, antagonize the receptor-mediated enzyme induction activity of 2,3,7,8-TCDD presumably via competitive displacement of 2,3,7,8-TCDD from the receptor protein. In contrast, coadministration of 2,3,7,8-TCDF and 2,3,7,8-TCDD gave additive enzyme induction responses. The identification of the 2,3,7,8-TCDD antagonists represents a new class of halogenated aryl hydrocarbons.

Validation of in vitro bioassays for 2,3,7,8-TCDD equivalents

Abstract The inducibility of aryl hydrocarbon hydroxylase (AHH) and ethoxyresorufin O-deethylase (EROD) in rat hepatoma H-4-II E cells culture has been utilized as a quantitative bioassay for polychlorinated dibenzo-p-dioxins (PCDDs) and dibenzofurans (PCDFs). There was an excellent linear correlation between the -log EC50 (in vitro enzyme induction) for 16 PCDD and PCDF congeners and their -log ED50 values for in vivo toxicity (body weight loss and thymic atrophy) in the rat. There was also a comparable linear correlation between the in vitro and in vivo AHH/EROD induction activities. Extraction of a fly ash sample with methylene chloride followed by cleanup gave a PCDD/PCDF fraction which contained a complex mixture of isomers and congeners. The total PCDD and PCDF levels were 3.83 and 5.52 ug/g fly ash respectively and 2,3,7,8-TCDD constituted 2–3% of the total tetrachlorodibenzo-p-dioxins. Using the in vitro enzyme induction bioassay procedure, the estimated 2,3,7,8-TCDD (equivalents) in this extract were 105 ng/g fly ash. The doseresponse in vivo induction of AHH was also determined and the estimated 2,3,7,8-TCDD (equivalents) in this extract were 75 ng/kg fly ash and this confirms the excellent correlation between the in vivo and in vitro bioassays.

Polychlorinated dibenzo-p-dioxins and dibenzofurans: Correlation between in vitro and in vivo structure-activity relationships (SARs)

Polychlorinated dibenzofurans (PCDFs) and dibenzo-p-dioxins (PCDDs) elicit a number of common biologic and toxic responses which are triggered by their initial binding to a cytosolic receptor protein. These effects include the induction of several cytochrome P-448 dependent monooxygenases (eg, aryl hydrocarbon hydroxylase, AHH), body weight loss and thymic atrophy. The dose-response effects of selected PCDFs on AHH induction in rat hepatoma H-4-II E cells and cytosolic receptor binding affinities have been determined. The results of these in vivo and in vitro studies demonstrate the remarkable effects of structure on the activity of PCDFs. A systematic study of each of the four different position for chlorine substitution in the dibenzofuran ring system showed that the toxic and biologic potencies of these compounds varied with respect to differential chlorine substitution at all four position, i.e. C-3(7) > C-2(8) >C-4(6) > C-1(9). In vitro SARs for PCDDs confirmed the importance of the lateral CI substituents and also showed that 1,2(or 6,7-) substituted PCDDs were more active than the corresponding 1,3-dichloro analogs. In addition, there were significant decreases in activity with increasing non-lateral CI substitution. The SARs for PCDFs were different from the PCDDs and this was directly related to the asymmetric structure of the former group of compounds.

Polychlorinated dibenzofurans; quantitative structure activity relationships

Polychlorinated dibenzofurans (PCDFs) are highly toxic industrial and combustion by-products which have been identified in diverse environmental matrices and have been implicated as etiologic agents in Yusho poisioning. The development of new synthetic routes in our laboratory has resulted in the unambiguous synthesis of over 50 purified PCDF congeners from their corresponding hydroxylated polychlorobiphenyl precursors. The effects of structure on the activity of PCDFs as inducers of aryl hydrocarbon hydroxylse (AHH) and as ligands for the Ah cytosolic receptor protein were determined. It was apparent that for both bioassays the most active compounds contained 4 lateral (2,3,7 and 8) chlorine atoms and that the degree of lateral substitution was a major structural determinant for activity. The receptor binding and AHH induction potencies (EC50) for the most active PCDF, 2,3,4,7,8-pentachlorodibenzofuran, were 1.5 × 10−8 M and 2.56 × 10−10 M respectively and these values were similar to the induction and receptor binding activities of 2,3,7,8-TCDD (1.0 × 10−8 M and 7.20 × 10−11 M). The in vitro structure-activity relationships (SARs) also indicated that substitution of C1 at the 4 and 6 positions in dibenzofuran gave PCDFs which were significantly more active than their corresponding isomers substituted at C-1 and C-9. The dose-response in vivo toxicities of several PCDFs were also determined in the rat using weight loss and thymic atrophy as toxic end-points. The in vivo quantitative structure-activity relationships for the PCDFs complemented the in vitro studies and confirmed that the more toxic PCDFs were substituted not only in the lateral positions but also at C-6 and C-4 of the dibenzofuran ring system.