S. Bandiera

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 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.

Bioanalysis of polychlorinated dibenzofuran and dibenzo-p-dioxin mixtures in fly ash

Abstract The biologic activities of the polychlorinated dibenzofuran and dibenzo- p -dioxin fractions from fly ash extracts are assessed by measuring their potencies as inducers of ethoxyresorufin 0-deethylase and aryl hydrocarbon hydroxylase in rat hepatoma H-4-II E cells and their binding affinities to the cytosolic receptor protein. The utility of these bioassays for the analysis of the toxic halogenated aryl hydrocarbons is discussed.

Hepta-, hexa-, tetra- and dichloronaphthalene congeners as inducers of hepatic microsomal drug-metabolizing enzymes.

Pretreatment of immature male Wistar rats with 1,2,3,4,5,6,7-hepta-, 1,2,3,4,5,6,8-hepta- and 1,2,3,4,5,6-hexachloronaphthalene resulted in the induction of several hepatic microsomal drug-metabolizing enzymes. The enzymic activities, reduced cytochrome P-450:CO and ethylisocyanide binding difference spectra and electrophoretic mobilities of the induced microsomal proteins were comparable to those observed after administration of the classical inducer of microsomal aryl hydrocarbon hydroxylase, 3-methylcholanthrene. The 1,2,3,4,5,6,7-heptachloronaphthalene congener, which is fully substituted in the lateral 2,3,6 and 7 positions, was more potent than the 1,2,3,4,5,6,8-hepta- and the 1,2,3,4,5,6-hexachloronaphthalene congeners which contain only 3 lateral chloro substituents. 1,2,3,4-Tetra- and several lower chlorinated naphthalenes were inactive as inducers of microsomal aryl hydrocarbon hydroxylase. The effects of structure on the induction activities of the polychlorinated naphthalenes were similar to those observed for other halogenated aryl hydrocarbons.

Octachloronaphthalene induction of hepatic microsomal aryl hydrocarbon hydroxylase activity in the immature male rat

Administration of octachloronaphthalene to immature male Wistar rats resulted in a dose-dependent increase in several enzymic, electrophoretic and spectral parameters associated with induction of the hepatic microsomal enzymes. Compared to corn-oil (control)treated animals octachloronaphthalene (150 mumol . kg-1) induced hepatic cytochrome P-450 (1.5-fold), benzo [alpha] pyrene hydroxylase (18-fold) and 4-chlorobiphenyl hydroxylase (18-fold) enzyme activities. In addition to increases in the relative peak intensities of the reduced microsomal cytochrome P-450 : CO and ethylisocyanide (EIC) difference spectra the peak maxima were observed at 448.5 and 452.2/428.0 nm, respectively. The effects of administering octachloronaphthalene to the rat were similar to those observed after pretreatment with 3-methylcholanthrene (MC) and electrophoresis of the induced microsomal proteins showed that both compounds enhance heme-staining peptides with comparable electrophoretic mobilities. Moreover coadministration of MC (3 x 100 mumol . kg-1) and octachloronaphthalene (2 x 150 mumol . kg-1) indicated that their inductive effects were not additive. It was concluded that octachloronaphthalene was an MC-type inducer of hepatic microsomal enzymes.

Antibodies as Probes of Cytochrome P450 Isozymes

Cytochrome P450*, an integral membrane protein, is widely distributed in many mammalian tissues, but is present in the highest concentration in hepatic endoplasmic reticulum. It functions as the terminal oxidase of an electron transport system that is involved in the metabolism of a large number of xenobiotics as well as endogenous substrates such as steroids, bile acids, fatty acids and prostaglandins (1). Although this enzyme system formerly was believed to function principally in detoxification, it is now known to metabolically activate many compounds to reactive metabolites that initiate toxic and carcinogenic events (2). Even more interesting is the observation that only certain optical isomers of bay-region diol epoxides of polycyclic aromatic hydrocarbons are carcinogenic, and that the cytochrome P450-dependent mixed function oxidase system preferentially catalyzes the formation of the optical isomer with the highest carcinogenic activity (3).

PCBs AS AHH INDUCERS

ABSTRACT The structure-activity rules for polychlorinated biphenyls (PCBs) as inducers of microsomal aryl hydrocarbon hydroxylase (AHH) activity have been determined by assessing the effects of synthetic PCB isomers and congeners on the immature male rat. PCBs which are AHH inducers must be substituted at both para positions, at least two meta positions, but not necessarily on the same ring; the introduction of one ortho chloro substituent into the nucleus of an AHH inducer does not eliminate this type of activity. The addition of a second ortho substituent eliminates the AHH activity in all the PCBs with exception of the 2,2′,3,3′,4,4′-hexa-, 2,2f,3′,4,4′,5-hexa-, 2,3,3′,4,4′,6-hexa-, 2,3,4,4′,5,6-hexa- and 2,2′,3,3′,4,4′,5-heptachlorobiphenyl congeners.

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.