The 2,3,7,8-tetrachlorodibenzo-p-dioxin Research Articles

2,3,7,8-Tetrachlorodibenzo- p-dioxin enhances responsiveness to post-ingestive satiety signals

The present study was designed to characterize the hypophagia that is a salient feature of the 2,3,7,8-tetrachlorodibenzo- p-dioxin (TCDD)-induced wasting syndrome. When TCDD-treated Long-Evans (L-E; dose 50 μg/kg) and Han-Wistar (H-W; 3000 μg/kg) rats were offered a simultaneous choice of three diets differing in their macronutrient composition, no selective aversion was seen to any of the varieties, although total energy intake decreased drastically and especially so in L-E rats. Further studies in H-W rats showed that TCDD treatment leads to a permanent retardation of weight gain accompanied by a decreased intake of chow and of a 10% sucrose solution, and to a reduced or unchanged consumption of water. In contrast, there was a progressive increase in saccharin drinking (when offered as the only choice) in TCDD-dosed rats with time. TCDD-treated animals also tended to consume a greater proportion of their daily feed intake during the daytime. These results imply that TCDD induces aversion to eating energy-providing food, irrespective of its type, and that TCDD exerts this at least in part by sensitizing the rats to post-ingestive satiety factors.

Effects of the food mutagens MeIQx and PhIP on the expression of cytochrome P450IA proteins in various tissues of male and female rats

The promutagenic 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx) and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) found in cooked food are converted to their active forms mainly by cytochrome P450 forms IA1 and IA2. By induction of these isoenzymes the food mutagens could thus influence their own rate of activation. Male and female Wistar rats were given MeIQx, PhIP, beta-naphthoflavone (BNF) or saline i.p. at 50 mg/kg body wt on three consecutive days. On the fourth day the rats were killed and lungs, kidneys, liver and intestines taken. The microsomal fraction from each organ was prepared as well as 9000 g supernatant from the liver. The induction of cytochrome P450IA was measured at the protein level by enzymatic assays (ethoxyresorufin-O-deethylation, Ames' mutagenicity test) and immunoassays (Western blot) and at the pretranslational level by RNA hybridization (Northern blot). The binding affinities of MeIQx, PhIP and 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) for the 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-receptor were studied by evaluation of the competition with 3H-labelled TCDD for specific binding. Ethoxyresorufin-O-deethylase (EROD) activity was significantly increased in the liver (males 2.1-fold, females 3.3-fold), kidneys (males 2.1-fold, females 1.8-fold) and lungs (males 4.3-fold, females 3-fold) of the MeIQx-treated rats. Furthermore, the levels of cytochrome P450IA proteins were increased in these animals. It was not possible, however, to detect the corresponding mRNA. In the case of the PhIP-treated animals a significantly increased EROD activity (2.7-fold) and an increased cytochrome P450IA protein level were seen only in the male lungs. Only a very weak TCDD-receptor affinity was observed for PhIP, whereas MeIQx or IQ did not appear to compete significantly with [3H]TCDD for binding to the TCDD-receptor. It is concluded that MeIQx is a weak inducer of cytochrome P450IA in several organs of the rat, while PhIP induced these isoenzymes only in the male lungs. More work is needed to clarify the mechanism(s) whereby this induction occurs.

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.

Immunopotentiation reverses the embryotoxic effect of serum from females with pregnancy loss

The 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) toxic equivalent quantity (TCDD-TEQ) approach was used successfully to predict lethal embryotoxicity in salmonids, but its applicability to sublethal effects of mixtures of organohalogenated compounds in other fish species is poorly known. The sublethal toxicity of two dioxin-like compounds (DLCs), 3,3′,4,4′-tetrachlorobiphenyl (PCB77) and 2,3,4,7,8-pentachlorodibenzofuran (2,3,4,7,8-PnCDF), two non-dioxin-like (NDL) polychlorinated biphenyls (PCBs), 2,2′,5,5′-tetrachlorobiphenyl (PCB52) and 2,3,3′,4′,6-pentachlorobiphenyl (PCB110), and of Aroclor 1254, a complex commercial mixture of PCBs, was assessed in Fundulus heteroclitus embryos exposed by intravitelline injection. At 16 days post-fertilization, the two DLCs and Aroclor 1254 altered prey capture ability in addition to inducing classical aryl hydrocarbon receptor-mediated responses: ethoxyresorufin-O-deethylase (EROD) induction, craniofacial deformities and reduction in body length. None of these responses was induced by the two NDL PCBs, at doses up to 5400 ng g−1 wet weight. Dose–response curves for prey capture ability for the 2 DLCs tested were not parallel to that of TCDD, violating a fundamental assumption for relative potency (ReP) estimation. Dose–response curves for EROD induction were parallel for 2,3,4,7,8-PnCDF and TCDD, but the ReP of 2,3,4,7,8-PnCDF for F. heteroclitus was 5-fold higher than the World Health Organization (WHO) fish toxic equivalent factor (TEF) based on embryolethality in salmonids. The chemically derived TCDD-TEQs of Aroclor 1254, calculated using 3,3′,4,4′,5-pentachlorobiphenyl (PCB126) concentrations and it ReP for F. heteroclitus, overestimated its potency to induce EROD activity possibly due to antagonistic interactions among PCBs. This study highlights the limitations of using TEFs based on salmonid toxicity data alone for risk assessment to other fish species. There is a need to assess the variability of RePs of DLCs in different species for a variety of endpoints and to better understand interactions between DLCs and other toxic chemicals.

Applications of the in vitro AHH induction bioassay for determining 2,3,7,8-TCDD equivalents: Pyrolyzed flame retardant mixtures

The pyrolysis of brominated flame retardants FireMaster 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 brominated dibenzofurans (PBDFs) and dibenzo-p-dioxins (PBDDs) as determined by gas chromatography-mass spectrometric analysis. The “2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) equivalents” for the pyrolyzed extracts were determined by measuring their aryl hydrocarbon hydroxylase (AHH) induction potencies in rat hepatoma H-4-II E cells in culture. The “2,3,7,8-TCDD equivalents” levels (ppm) for each of the pyrolyzed flame retardant samples was: 174–194, 480–1400, 2140–4680, 6740–8780 and 3920–5260 for FireMaster 300 BA, FireMaster BP-6, Bromkal 70 DE, Bromkal 70-5 DE and Bromkal G1 [note: the range of equivalents was derived from the values obtained from the AHH and ethoxyresorufin O-deethylase (EROD) enzyme assays]. The “2,3,7,8-TCDD equivalents” in the pyrolyzed FireMaster BP-6 and Bromkal 70-5 DE samples were determined in in vivo studies (rat); there was an excellent correlation between the in vivo and in vitro data and these results further support to the utility of the in vitro induction bioassay for quantitatively determining “2,3,7,8-TCDD equivalents” for toxic halogenated aromatic mixtures.

Polyacrylamide concentration gradient gel electrophoresis of the 2,3,7,8-tetrachlorodibenzo- p-dioxin (TCDD) receptor in rat liver cytosol

An assay based on polyacrylamide concentration gradient gel electrophoresis for the 2,3,7,8-tetrachlorodibenzo- p-dioxin (TCDD) receptor in rat liver cytosol was developed. [ 3H]TCDD-labeled cytosol was treated with dextran-coated charcoal and electrophoresed for 3000 Vh in polyacrylamide concentration gradient gels ( T = 2.5–20%). A Tris-borate running buffer (pH 8.35) without salt and detergents was used. When the polyacrylamide concentration gradient was concave a linear calibration curve of log molecular weight vs. migration distance was obtained for the standard proteins used. Under these conditions the M Γ value of the TCDD receptor from rat liver cytosol was estimated to be 316 000 ±27 000 before, and 246 000 ± 12 000 after limited proteolysis. The B max for the specific binding of [ 3H]TCDD was between 35 and 43 fmol/mg cytosolic protein and the K d of the binding ranged from 0.8 to 1.1 nM, as judged by saturation and Scatchard analysis.

Clinical Laboratory Manifestations of Exposure to Dioxin in Children

Following a major environmental accident near Seveso, Italy, on July 10, 1976, we attempted to determine if the 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) released into the atmosphere had any effect on the liver function and lipid metabolism of exposed children. From July 1976 to June 1982, we analyzed the results of more than 4500 laboratory tests (alanine aminotransferase, aspartate aminotransferase, gamma-glutamyltransferase, alkaline phosphatase, cholesterol, and triglycerides in plasma and delta-aminolevulinic acid in urine) in a population of about 1500 children aged 6 to 10 years at the moment of the accident. The children exposed to the highest concentration of TCDD showed alterations in serum gamma-glutamyltransferase and alanine aminotransferase activity compared with the control group. These differences were restricted to values inside limits set from the lower end of the normal range to slightly above it. The observed abnormalities were slight and disappeared with time.

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 ED 50 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 EC 50 values for AHH or EROD induction in cell culture and the -log ED 50 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.

Elevation of 2,3,7,8-tetrachlorodibenzo- p-dioxin (TCDD) polychlorinated biphenyls : Structure-activity relationships

Administration of the commercial polychlorinated biphenyl (PCB) Aroclor 1254 to immature male Wistar rats resulted in increased levels (80–110%) of the 2,3,7,8-tetrachlorodibenzo- p-dioxin (TCDD) hepatic cytosolic receptor protein which remained elevated for 14 days. The effects of structure on the activity of individual PCB congeners to modulate hepatic cytosolic receptor levels were compared to the structure-activity relationships (SARs) which have been developed previously for PCBs as inducers of hepatic microsomal monooxygenases. 3,3′,4,4′-Tetra- and 3,3′,4,4′,5-pentachlorobiphenyl induced the cytochrome P-448-dependent monooxygenase, ethoxyresorufin O-deethylase (EROD), and resembled 3-methylcholanthrene in their mode of monooxygenase enzyme induction. These congeners also bound to the receptor protein; however, neither compound increased hepatic cytosolic receptor protein levels. Several PCB congeners which exhibit low binding affinities for the cytosolic receptor protein resembled phenobarbitone (PB) in their mode of monooxygenase enzyme induction and, like PB, elevated cytosolic receptor protein levels. Nevertheless, a comparison of the time course of monooxygenase enzyme induction and receptor protein elevation by 2,2′,4,4′,5,5′-hexachlorobiphenyl and PB illustrated significant differences in their activities. PB-mediated elevation of receptor levels was maximized 24 hr after the last dose, and 48 hr later the receptor levels decreased to control values. In contrast, 5 days after administration of a single dose of 2,2′,4,4′,5,5′-hexachlorobiphenyl (300 μmoles/ kg) the receptor levels were elevated significantly, and these increased levels (205-127% increases over control) persisted for 14 days. There was no correlation between increased levels of hepatic receptor protein and the induction of the cytochrome P-450-dependent monooxygenases, aldrin epoxidase or 4-dimethylaminoantipyrine N-demethylase. Two PCBs, 2,3,3′,4,4′,5- and 2,2′,3,4,4′,5-hexachloro-biphenyl, which resembled Aroclor 1254 in their mode of monooxygenase enzyme induction, also elevated hepatic receptor protein levels but were less active than the PB-type inducers. Thus, the SARs developed for PCBs which elevate cytosolic receptor levels demonstrate that the most active compounds exhibit the lowest affinity for the receptor protein and do not induce EROD. In contrast, the more toxic PCB congeners which are approximate isostereomers of 2,3,7,8-TCDD both induced EROD and bound with high affinity to the receptor protein but did not increase hepatic cytosolic receptor protein levels.

A hydroxylapatite microassay for receptor binding of 2,3,7,8-tetrachlorodibenzo- p-dioxin and 3-methylcholanthrene in various target tissues

A “batch” hydroxylapatite assay for the 2,3,7,8-tetrachlorodibenzo- p-dioxin (TCDD) receptor that does not require detergents is described. The receptor could be assayed in rat target tissues using either of the cytochrome P 1-450 inducers [ 3H]TCDD or [ 3H]3-methylcholanthrene as radioligands. A phosphate buffer washing procedure was developed on the basis of chromatographic data and optimized to separate nonspecifically and specifically bound ligand. The assay was characterized with respect to washing efficiency, binding specificity, competition, adsorption time, amount of hydroxylapatite required to bind receptor complexes, sensitivity, and effects of detergents. Equilibrium binding parameters were determined. Receptor extracted with phosphate from hydroxylapatite was analyzed on sucrose gradients and was found to exhibit the same sedimentation properties as the receptor in crude cytosol. Furthermore, the applicability of the assay has been demonstrated in cytosolic preparations from three different target tissues: liver, lung, and thymus.

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 ED 50 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 ED 50 for the most active PCDF, 2,3,4,7,8-pentachlorodibenzofuran was 1.5 × 10 −8 M which was only slightly less active than 2,3,7,8-TCDD (1.0 × 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. 1,2,4,5,7-/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 PCDFFs 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.

Short-term bioassays of nitro derivatives of benzo[a]pyrene and perylene.

Several nitroarenes derived from benzo[a]pyrene and perylene and the parent hydrocarbons have been assayed for mutagenicity in the Salmonella microsome test and for affinity for the 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-receptor protein in rat liver cytosol. 1- and 3-nitrobenzo[a]pyrene are mutagenic in the absence of S9 and have a response pattern in several Salmonella strains resembling that shown by 1-nitropyrene. 6-Nitrobenzo[a]pyrene, 3-nitroperylene as well as the parent unsubstituted hydrocarbons require S9 for activation. The 3,6- and 3,7-dinitroperylenes and a mixture of 3,9-/3,10-dinitroperylene are all mutagenic in the absence of S9. The response pattern of 3,9-/3,10-dinitroperylene resembles that shown by 1,6- and 1,8-dinitropyrenes. The nitro derivatives which are active in the absence of S9 are all inactivated by the addition of an incomplete S9 lacking NADP but activity is regained in the presence of the complete S9 for 1-and 3-nitrobenzo[a]pyrene and 3,6-dinitroperylene, showing that these compounds are also activated by S9 enzymes. Benzo[a]pyrene, 1- and 3-nitrobenzo[a]pyrene, 3-nitroperylene and the mixture of 3,9-/3,10-dinitroperylene have a high affinity for the TCDD-receptor protein whereas the affinity is low or below the detection level for the other compounds. These results are in good agreement with known structural requirements for receptor binding.