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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 in vitro induction of the cytochrome P1-450-dependent monooxygenases, aryl hydrocarbon hydroxylase (AHH) or ethoxyresorufin O-deethylase (EROD) by 2,3,7,8-TCDD and related toxic halogenated aryl hydrocarbons in rat hepatoma H-4-II E cells has been developed as a short term quantitative bioassay for these toxic chemicals. There was a linear correlation between the -log EC50 (in vitro) AHH induction vs the -log ED50 (in vivo) for body weight loss, thymic atrophy, hepatic AHH and EROD induction in the rat for several polychlorinated biphenyl, dibenzo-p-dioxin and dibenzofuran congeners and mixtures. These data clearly support the utility of the in vitro AHH induction assay as a short term test system for quantitating the “toxic or 2,3,7,8-TCDD equivalents” in an extract containing toxic halogenated aromatics. The bioassay method is rapid, relatively accurate and much more cost effective than conventional analytical methods such as gas-chromatography-mass spectrometry from which it is difficult to determine the levels of 2,3,7,8-TCDD equivalents in specific analytes.

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.

The photolysis products and reaction quantum yields from a number of simple halonaphthalenes have been determined. Fluoronaphthalenes react from a singlet state to yield substitution products in nucleophilic solvents. Chloronaphthalenes and 1-bromonaphthalene afford mainly radical products (naphthalene and binaphthyls) through a triplet state, though some photonucleophilic substitution occurs with chloronaphthalene. Attempts at quenching lead to abnormal behavior, in that enhanced quantum yields for reaction are found with several potential triplet quenchers. Electron transfer processes are proposed to rationalize the observed products.

The synthesis of all possible laterally-substituted polybrominated biphenyl (PBB) congeners containing two para bromines is described. Using enzymic, electrophoretic and ligand-binding assays that distinguish between phenobarbitone(PB)- and 3-methylcholanthrene(MC)-type inducers, the synthetic PBBs were evaluated as inducers of liver microsomal drug-metabolizing enzymes in the immature male Wistar rat. 4,4'-Dibromobiphenyl resembled PB in its mode of induction whereas all the meta-brominated derivatives of 4,4'-dibromobiphenyl, namely 3,4,4'-tri, 3,4,4',5-tetra-, 3,3', 4,4'-tetra-, 3,3',4,4',5-penta- and 3,3',4,4',5,5'-hexabromobiphenyl, resembled MC in their mode of induction. The results obtained with 3,4,4'-tribromobiphenyl demonstrate that, in contrast to the polychlorinated biphenyls (PCBs), a single meta halogen substituent is sufficient to abolish the PB-type characteristics of 4,4'-dibromobiphenyl and convert it to a strictly MC-type inducer. PBBs which induce AHH activity must be substituted at both para positions and at one, two, three or four meta positions. Ortho-substitution of PBBs which contain only lateral bromine groups may also give compounds which are aryl hydrocarbon hydroxylase (AHH) inducers. One of the MC-type PBBs, namely 3,3',4,4'-tetrabromobiphenyl, which has been tentatively identified in the commercial PBB mixture, fireMaster BP-6, was at least 50 times more potent as an inducer of AHH activity than the commercial PBB mixture. The induction of AHH by 3,3',4,4'-tetrabromobiphenyl was accompanied by a dose-dependent decrease in both thymus and spleen weights. The thymus and/or spleen weights were decreased in rats treated with the other MC-type PBBs which further supports the correlation between the toxicity of the PBBs and their ability to induce AHH.

The activities of several individual polychlorinated biphenyls (PCBs) and dibenzofurans (PCDFs) and several environmentally significant reconstituted mixtures of these compounds as inducers of aryl hydrocarbon hydroxylase (AHH) and ethoxyresorufin O-deethylase (EROD) in rat hepatoma H-4-II E cells were determined. The observed AHH and EROD induction EC50S for the mixtures were compared with the calculated values, which were based on the summation of the relative per cent contributions of the individual components of the reconstituted PCB and PCDF mixtures. The results show that the differences between the observed and calculated EC50s for these mixtures were minimal or not significant and the data supports the use of the rat hepatoma H-4-II E cell system as a bioassay for toxic halogenated aryl hydrocarbons.

Several investigators have reported the finding of polycystic kidney disease (PKD) at autopsy in patients who had undergone long-term hemodialysis for renal failure due to causes other than PKD. We initiated studies to determine whether the drugs or chemicals to which patients on dialysis are exposed could be responsible for these cystic changes. Adult rats were tube fed chemical residues from an artificial kidney or phthalate esters [di(2-ethylhexyl) phthalate, DEHP], which are a main component of these plastic kidneys or a control solution. Rats receiving DEHP or residues showed a significantly higher incidence of focal cysts when compared to controls. Rats receiving DEHP developed a significant decrease in kidney function as demonstrated by creatinine clearance, and these animals had the highest tissue levels of measureable DEHP. We postulate that patients receiving long-term dialysis may acquire PKD secondary to their exposure to chemicals leached from artificial kidneys.

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.

A comparison of the molecular properties of the male Long-Evans rat and male C57BL/ 6 mouse hepatic cytosolic aryl hydrocarbon (Ah) receptor complex was determined using 2,3,7,8-[ 3 H]tetrachlorodibenzo- p -dioxin (TCDD) and 2,3,7,8-[ 3 H]tetrachlorodibenzofuran (TCDF) as radioligands. In low salt buffer, the sedimentation coefficients, Stokes radii, relative molecular masses, frictional ratios, axial ratios and gel permeation chromatographic properties of the rat receptor complexes were ligand independent. In contrast, there were several ligand-dependent differences in the mouse Ah receptor complexes formed after incubation in low salt buffer and these include: sucrose density gradient analysis of the 2,3,7,8-[ 3 H]TCDF receptor complex gave a 9.5 S specifically bound peak and a 2.6 S nonspecifically bound peak whereas the corresponding 2,3,7,8-[ 3 H]TCDD receptor complex gave a single 9.6 S specifically bound peak; sucrose density gradient analysis of the two major peaks eluted from a Sephacryl S-300 column chromatographie separation of the 2,3,7,8-[ 3 H]TCDF receptor complex gave two specifically bound peaks at 9.2 and 5.1 S. The molecular properties of the rat hepatic cytosolic receptor complexes incubated in high salt (0.4 m KCl) buffer were ligand independent with one exception, namely the significant difference in the sedimentation coefficient of the specifically bound disaggregated 2,3,7,8-[ 3 H]TCDD receptor complex (6.8 S) and the corresponding 2,3,7,8-[ 3 H]TCDF receptor complex (5.0 S). The major ligand-dependent differences in the mouse receptor complexes incubated in high salt (0.4 m KCl) were associated with the sedimentation coefficients of the complexes derived after direct incubation and after gel permeation chromatography. For example, both ligands gave two specifically bound complexes after chromatography on Sephacryl S-300 column and centrifugation of these fractions gave both the ~9 and ~5 S peaks; this suggested that there was some equilibration between the aggregated and disaggregated receptor complexes. The behavior of the 2,3,7,8-[ 3 H]TCDF mouse receptor complex was similar after incubation in low or high salt buffer except that sucrose density gradient analysis of the gel permeation Chromatographic fractions gave an additional specifically bound peak which sedimented at 7.2 S. These studies demonstrate that the molecular properties of the Ah receptor were dependent on the source of the cytosolic receptor preparation, the ionic strength of the incubation media, and the structure of the radioligand.

Hymenoxon and helenalin are toxic sesquiterpene lactones present in the toxic range plants Hymenoxys odorata and Helenium microcephalum. Helenalin (25 mg/kg) or hymenoxon (30 mg/kg) administered to immature male ICR mice caused a rapid decrease in hepatic glutathione levels and were lethally toxic to greater than 60% of the animals within 6 d. L-2-Oxothiazolidine 4-carboxylate (OTC), a compound that elevates cellular glutathione levels, administered to mice 6 or 12 h before either helenalin or hymenoxon protected against hepatic glutathione depletion and the lethal toxicity of these toxins. OTC administered at the same time as the sesquiterpene lactones was not protective, suggesting that the critical events against which glutathione is protective occur within the first 6 h. In primary rat hepatocyte cultures, hymenoxon and helenalin (4-16 microM) caused a rapid lethal injury as determined by the release of lactate dehydrogenase. Cotreatment of cultures with N-acetylcysteine at high concentrations (4 mM) afforded significant protection against lethal injury by both toxins. In contrast, BCNU, which inhibits glutathione reductase, or diethylmaleate, which depletes hepatocellular glutathione, potentiated the hepatotoxicity of helenalin and hymenoxon in monolayer rat hepatocytes. These studies suggest that the in vivo and in vitro toxicity of hymenoxon and helenalin is strongly dependent on hepatic glutathione levels, which hymenoxon and helenalin rapidly deplete at very low concentrations.

The effects of structure on the activity of 27 polychlorinated dibenzo-p-dioxin (PCDD), dibenzofuran (PCDF), biphenyl (PCB) and polybrominated dibenzo-p-dioxin (PBDD) congeners has been determined in the immature male Wistar rat [body weight loss, thymic atrophy, hepatic microsomal aryl hydrocarbon hydroxylase (AHH) and ethoxyresorufin O-deethylase (EROD)] and in rat hepatoma H-4-II E cells in culture (AHH and EROD induction). There was a good linear correlation between the -log EC50 values for AHH induction (in vitro) and the in vivo -log ED50 values for AHH induction (r=0.85), body weight loss (r=0.93) and thymic atrophy (r=0.92) for the 27 compounds investigated in this study. Comparable in vivo studies have also been carried out using several PCDD and PCDF congeners (2,3,7,8-, 1,3,7,8- and 1,2,4,7,8-substituted PCDDs; 2,3,4,7,8-, 1,2,3,7,8-, 2,3,4,7,9- and 1,2,3,7,9-substituted PCDFs) using the guinea pig as the model. There were excellent linear correlations (r > 0.92) between the -log EC50 (in vitro AHH induction) vs. the -log ED50 (in vivo responses) and the -log ED50 (guinea pig in vivo data) vs. -log ED50 (rat in vivo data). These results further validate the utility of the in vitro AHH induction assay for quantitatively estimating the potential toxicity of PCB, PCDF and PCDD mixtures in environmental extracts. This assay can be carried out rapidly, is relatively inexpensive and utilizes the established (and stable) rat hepatoma H-4-II E cells in culture which can detect 20–50 pg of 2,3,7,8-TCDD equivalents per plate.

A comparison of the molecular properties of the rat and mouse hepatic cytosolic Ah receptor complex was determined using [3H]-2,3,7,8-tetrachloro-p-dioxin (TCDD) and [3H]-2,3,7,8-tetrachlorodibenzofuran (TCDF) as radioligands. In low salt buffer, the sedimentation coefficient, Stokes radii, relative molecular masses, frictional ratios, axial ratios and gel permeation chromatographic properties of the rat receptor complexes were ligand-independent. In contrast, there were several ligand-dependent differences in the mouse Ah receptor complexes formed after incubation in low salt buffer and these included the following: sucrose density gradient analysis of the [3]-2,3,7,8-TCDF receptor complex gave a 9.5 S specifically bound peak and a 2.6 S non-specifically bound peak whereas the corresponding [3H]-2,3,7,8-TCDD receptor complex gave a single 9.6 S specifically bound peak: sucrose density gradient analysis of the two major peaks eluted from Sephacryl S-300 column after chromatographic separation of the [3H]-2,3,7,8-TCDF receptor complex gave two specifically-bound peaks at 9.2 and 5.1 S. The molecular properties of the rat hepatic cytosolic receptor complexes incubated in high salt (0.4 M KCl) buffer were ligand-independent with one exception, namely the significant difference in the sedimentation coefficient of the specifically bound disaggregated [3H]-2,3,7,8-TCDD receptor complex (6.8 S) and the corresponding [3H]-2,3,7,8-TCDF receptor complex (5.0 S). The major ligand-dependent differences in the mouse receptor complexes incubated in high salt (0.4 M) were associated with the sedimentation coefficients of the complexes derived after direct incubation and after gel permeation chromatography. For example, both ligands gave two specifically bound complexes after chromatography on Sephacryl S-300 column and centrifugation of these fractions gave both the ∼9S and ∼5S peaks: this indicated that there was equilibration between the aggregated and disaggregated receptor complexes. The behavior of the [3H]-2,3,7,8-TCDF mouse receptor complex was similar after incubation in low or high salt buffer except that sucrose density gradient analysis of the gel permeation chromatographic fraction gave an additional specifically-bound peak which sedimented at 7.2 S. These studies demonstrate that the molecular properties of the Ah receptor are dependent on the source of the cytosolic receptor preparation, the ionic strength of the incubation media and the structure of the radioligand.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) and 1,2,3,7,8-pentachlorodibenzo-p-dioxin (PCDD) were administered by oral gavage [1 ug/kg/week in corn oil (5 ml/kg)] to female Sprague-Dawley rats for up to six months. Liver and kidney DNA were isolated and the formation of DNA adducts in liver and kidney were determined by the highly sensitive 32p-postlabeling assay procedure. No exposure-related spots associated with TCDD or PCDD covalent DNA adducts were noted on the chromatograms of kidney or liver DNA nucleotides from the rats exposed to the toxins for 2 and 6 months. Corn oil treated animals exhibited the characteristic tissue- and age-specific patterns of 32p-labeled I-spots (Randerath, Reddy and Disher, Carcinogenesis 7, 1615, 1986) which are associated with specific DNA modifications of unknown function. Treatment with either PCDD or TCDD resulted in significant and substantial reduction of the levels of the I-compounds in the liver but not the kidney DNA. Reductions in the amounts of individual hepatic I-compounds ranged from 45–65 and 40–80% in rats treated with TCDD for 2 and 6 months respectively. The effects of PCDD were less marked than those observed for TCDD.

Analysis of male Sprague--Dawley rat hepatic cytosol from two commercial animal laboratories for the polycyclic aromatic hydrocarbon (PAH) 4-5S binding protein showed that in one group of animals no 4-5S protein was detectable (-4S) whereas the levels of this protein were 208 +/- 57 fmol/mg cytosolic protein in the +4S rats. The role of the 4-5S binding protein in the transregulation of the cytochrome P-450-dependent monooxygenase, aryl hydrocarbon hydroxylase (AHH), was therefore investigated in the -4S and +4S Sprague-Dawley rats. The dose-response curves for the induction of hepatic microsomal AHH by 3-methylcholanthrene (MC) were indistinguishable in both +4S and -4S rats and comparable results were observed for 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) as an inducer. Both MC and TCDD exhibit high binding affinities for the aryl hydrocarbon (Ah) 8-9S receptor protein, whereas MC but not TCDD bound with high affinity to the 4-5S binding protein. Benzo[a]pyrene (B[a]P) binds with moderate affinity to both the Ah receptor and 4-5S binding protein and induces AHH in both -4S and +4S rats. Perylene binds with moderate affinity to the 4-5S binding protein but does not interact with the Ah receptor. This PAH was inactive as an inducer of AHH in +4S and -4S Sprague-Dawley rats. These results show that there was a correlation between the Ah receptor binding affinities of MC, B[a]P and perylene and their potencies as AHH inducers in Sprague-Dawley rats, and this corresponds to previous correlations for the induction of AHH in rat hepatoma H-4-II E cells in culture. In contrast no such correlations existed between the AHH induction potencies of these polynuclear aromatic hydrocarbons and their affinities for the 4-5S binding protein. These data, coupled with the fact that the absence of the 4-5S binding protein in the -4S Sprague-Dawley rats did not affect AHH inducibility by MC, B[a]P or perylene, suggests that the 4-5S binding protein does not play a role in the transregulation of cytochrome P-4501A1 in the rat or rat hepatoma cells in culture.

The mechanism of action of PCDDs and PCDFs has been extensively investigated and the results from a multitude of studies support the role of a receptor-mediated process. Initial binding of the PCDD or PCDF ligand to the aryl hydrocarbon (Ah) receptor is necessary but not sufficient for eliciting the diverse biologic and toxic responses elicited by toxic PCDDs and PCDFs. Although the interspecies potencies of PCDDs/PCDFs (eg. 2,3,7,8-TCDD) may vary widely, the structure-activity relationships (SARs) observed for Ah receptor-mediated in vitro and in vivo responses are comparable. This observation leads to 2 important mechanism-based conclusions; namely, 2,3,7,8-TCDD equivalents values (or toxic equivalency factors) can be utilized for risk assessment of individual PCDD and PCDF congeners and simple in vitro (eg., AHH induction in rat hepatoma cells) or in vivo bioassays can be used for risk assessment of PCDD/PCDF mixtures.

Administration of several persistent PCB congeners to male Wistar rats demonstrated a structure-dependent elevation of hepatic cytosolic 2,3,7,8-TCDD receptor levels. 2,2′,4,4′5,5′-Hexachlorobiphenyl and several related compounds which exhibit low affinity for the receptor protein increased receptor levels to over 300% compared to the control animals. Aroclor 1254 and 2,3,3′,4,4′,5′-hexachlorobiphenyl both bind with moderate affinity to the receptor protein (10 uM) and effect some elevation of receptor levels. In contrast, 3,3′,4,4′,5-pentachlorobiphenyl, a toxic PCB with high in vitro binding affinity (10 uM) does not alter hepatic receptor levels. Time course studies, demonstrated that 2,2′,4,4′,5,5′-hexachlorobiphenyl elevated hepatic cytosolic receptor levels for at least 14 days with the maximum elevation occurring 7 days after initial exposure to this PCB congener. In contrast, dose and time-response studies with hexachlorobenzene, several DDT analogs and heptachlor expoxide gave variable results with respect to hepatic 2,3,7,8-TCDD receptor modulation. For the PCBs, it was apparent that the most active congeners exhibited low affinity for the receptor and were phenobarbitone (PB)-like inducers of microsomal monooxygenases. Although all the organochlorine insecticides which were tested also were PB-type monooxygenase enzyme inducers only the DDT analogs modulated hepatic receptor levels.

Hexachlorobenzene (400 μimol/kg) induced rat hepatic microsomal aryl hydrocarbon hydroxylase (AHH) and ethoxyresorufin O‐deethylase (EROD), however at dose levels up to 3000 μmol/kg, the induced enzyme levels were less than 50% of the maximum induced activities caused by either 3‐methylcholanthrene (MC) or 2,3,7,8‐tetrachlorodibenzo‐p‐dioxin (TCDD). HCB did not significantly induce these monooxygenases in the thymus or lung but some time‐dependent induction of AHH and EROD was observed in the rat kidney. In contrast, MC induced microsomal AHH and EROD in the rat liver, lung and kidney but not the thymus. Cotreatment of HCB (400 μmol/kg) plus 2,3,7,8‐TCDD (10 or 30 μg/kg) resulted in some small but significant dose‐ and organ‐dependent modulation of the activity of 2,3,7,8‐TCDD as an inducer of AHH and EROD. The 30 μg/kg dose of 2,3,7,8‐TCDD was toxic and this may account for the lower induced enzyme levels observed in the kidney and thymus of animals cotreated with HCB plus 2,3,7,8‐TCDD compared ...

Previous studies have demonstrated that 1,3,6,8-tetrachlorodibenzofuran (TCDF) inhibited the induction of aryl hydrocarbon hydroxylase (AHH) and ethoxyresorufin O-deethylase (EROD) in rat hepatoma H-4-II E cells in culture. 1,3,6,8-TCDF did not induce hepatic microsomal AHH or EROD at doses up to 800 umol/kg in DBA/2 mice and 200 umol/kg in C57BL/6 mice. Administration of 1,3,6,8-TCDF to the latter strain of mice resulted in induction of AHH and EROD (< 25% of maximal induction activity). Cotreatment of mDBA/2 mice with 2,3,7,8-TCDD (200 nmol/kg) and with either 50, 200 or 800 umol/kg of 1,3,6,8-TCDF resulted in 13, 39 and 18% inhibition of AHH induction and 17, 34 and 21% inhibition of EROD induction respectively. Cotreatment of the more responsive C57BL/6 mice with 2,3,7,8-TCDD (15 nmol/kg) and 1,3,6,8-TCDF (10, 50, 100, 200 or 500 umol/kg) resulted in significant inhibition (17%) of AHH and EROD induction only at a 200 umol/kg dose level. It was evident that the apparent window of antagonism for 1,3,6,8-TCDF was strain dependent.

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.

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.

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 ED 50 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 EC 50 (in vitro AHH induction) vs. the in vivo -log ED 50 (thymic atrophy) and -log ED 50 (body wt loss) correlation coefficient, r = 0.97 for all 2 correlations).

The influences of in vivo treatment with two pure PCB congeners, 2,2',4,4',5,5'-hexachlorobiphenyl (HCBP) and 3,3',4,4'-tetrachlorobiphenyl (TCBP), on the lethal cytotoxicity of bromobenzene and acetaminophen were examined in short-term primary cultures of isolated rat hepatocytes. Lethal injury was measured by release of lactate dehydrogenase (LDH) into culture medium after 20 hr exposure to the hepatotoxins. The HCBP, a PB-type inducer of cytochrome P-450, resembled phenobarbitone (PB) in its ability to increase susceptibility of hepatocytes to bromobenzene (0.5 to 1.6 mM) and acetaminophen (1 to 16 mM). This induced sensitivity was consistently inhibited by SKF-525-A (10 microM) but not alpha-naphthoflavone (ANF, 10 microM) in culture. The 3,3',4,4'-TCPB, a 3-MC-type inducer of cytochrome P-450, resembled 3-methylcholanthrene (3-MC) in its inability to induce susceptibility to bromobenzene. TCBP and 3-MC each increased (20- to 30-fold) cytotoxicity of acetaminophen by a mechanism substantially inhibitable by ANF but not SKF-525-A. These results demonstrate that categorizing pure PCB isomers and congeners into groups according to their different induction capabilities is predictive for their ability to modulate acute hepatocellular necrosis by bromobenzene and acetaminophen.

Administration of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD, 20 micrograms/kg) to pregnant C57BL/6J mice (on day 10) resulted in 62% fetuses with cleft palate per litter without any observable maternal toxicity. In contrast, Aroclor 1254 administered at a dose of 750 mumol/kg was not teratogenic. Cotreatment of the pregnant mice with both Aroclor 1254 (244 mg/kg) and 2,3,7,8-TCDD (20 micrograms/kg) resulted in an 8.2% incidence of cleft palate per litter. In contrast, Aroclor 1254 did not afford any protection from the teratogenicity of dexamethasone in C57BL/6J mice. Previous studies have shown that Aroclor 1254 can act as a partial antagonist of the microsomal enzyme induction and immunotoxic effects of 2,3,7,8-TCDD in C57BL/6J mice and this paper demonstrates that the commercial polychlorinated biphenyl mixture also antagonizes 2,3,7,8-TCDD-mediated teratogenicity in this strain of mice.

3,3',4,4'-Tetrabromobiphenyl is a minor component of commercial polybrominated biphenyl (PBB) mixture fireMaster BP-6 and is a potent inducer of aryl hydrocarbon hydroxylase (AHH). A single ip dose of 3,3',4,4'-tetrabromobiphenyl (150 mumol/kg) caused significant reduction in the growth rate in the immature male Wistar rat, as well as pale enlarged livers and marked reduction in thymus size. Under light microscopy, hepatocytes were enlarged and vacuolated. The vacuoles, which were most prominent in the midzonal region of the lobule, corresponded to fat droplets in oil-red-O-stained sections. The thymus, especially the cortex, was markedly depleted of lymphocytes. Neither the reduced growth, altered organ weights nor the histopathology was reversed for the duration of the study by the coadministration of the antioxidants butylated hydroxy anisole (BHA), butylated hydroxy toluene (BHT), or vitamin E. Vitamin E did, however counter the negative effect of 3,3',4,4'-tetrabromobiphenyl on growth during the first 5 d of the study. 2,2',5,5'-Tetrabromobiphenyl, also a minor component of fireMaster BP-6, is a weak phenobarbital-type inducer of cytochrome P-450. When administered at the same dose, 2,2',5,5'-tetrabromobiphenyl did not elicit any observed toxic effects. These data confirm the correlation between AHH induction and toxicity for these PBBs and suggest that 3,3',4,4'-tetrabromobiphenyl may significantly contribute to the toxicity of fireMaster BP-6. Although there is evidence that polychlorinated biphenyls, and perhaps 2,3,7,8-tetrachlorodibenzo-p-dioxin, exert certain toxic effects via a lipid peroxidation mechanism, the toxic changes measured during this study were not reversed by the administration of the antioxidants.

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.

Halogenated hydrocarbon insecticides and polychlorinated biphenyl (PCB) mixtures are routinely detected as residues in human adipose tissue, serum, and milk. Based on average values observed in analytical studies, reconstituted halogenated hydrocarbon pesticide mixtures and PCB mixtures were prepared and administered to immature male Wistar rats. The mixtures were administered at dose levels which approximate the concentrations which would be absorbed by an infant suckling for 180 days (low dose, L), and at three higher dose levels (2 × L, 10 × L, and 100 × L). The pesticide mixture contained isomeric hexachlorocyclohexanes, dieldrin, heptachlor epoxide, oxychlordane, trans-nonachlor, hexachlorobenzene, 1,1,1-trichloro-2,2-bis(p-chlorophenyl)ethane, 1,1-dichloro-2,2-bis(p-chlorophenyl)ethane, and 1,1-dichloro-2,2-bis(p-chlorophenyl)ethylene; the reconstituted PCB mixture contained 13 of the major congeners which have been identified in human milk samples. Administration of the L dose level of the pesticide (0.95 mg/kg), PCB (0.45 mg/kg), and pesticide plus PCB mixture (0.95 + 0.45 mg/kg, respectively) in corn oil on days 1 and 3 did not significantly alter hepatic drug-metabolizing enzyme activities or elicit any observable pathological damage 6 days after the first exposure. In contrast, administration of the higher dose levels of this mixture elicited a dose-dependent induction of several hepatic drug-metabolizing enzymes. Moreover, despite the short duration of exposure to these chemicals, the rats treated with the higher doses (10 × L and 100 × L) of these mixtures exhibited mild alterations in thyroid architecture, changes in hepatocellular nuclei including variations in chromatin distribution, vesiculation of larger nuclei, and frequent appearance of pyknotic shrunken nuclei. In addition there were changes in hepatocellular cytoplasm organization including vacuolations and mild zonal variations in volume. The results clearly indicate that relatively low doses of the pesticides and pollutant mixtures commonly found in human breast milk can elicit significant biologic and toxic effects in the immature male Wistar rat.

The condensation of 2,6-dichloroaniline and 2,5-dichlorophenol gave 2-hydroxy-2′,3,6,6′-tetrachlorobiphenyl which was readily cyclized to 1,4,9-trichlorodibenzofuran (TrCDF). 1,4,9-TrCDF was converted into [1,4,9-3H3]-dibenzofuran by catalytic exchange with tritium gas. The tritiated dibenzofuran was dissolved in methylene chloride and chlorine gas was bubbled into the solution for 20 minutes to give the following PCDF congeners which were separated and purified by HPLC; [3H]-2,3,7,8-tetrachlorodibenzofuran (TCDF, 57 Ci/mmol), [3H]-1,2,3,7,8-pentachlorodibenzofuran (PeCDF, 34 Ci/mmol) and [3H]-1,2,3,6,7,8-/1,2,3,4,7,8-hexachlorodibenzofuran (HCDF, 32.5 Ci/mmol). The dose-dependent binding of the new radioligands with different concentrations of cytosolic receptor protein was saturable. Scatchard plot analysis of the binding isotherms gave Kd values of 1.3 ± 0.07 (2), 5.9 ± 1.2 (2), 2.3 ± 1.5 (2) nM respectively for radiolabeled 2,3,7,8-TCDF, 1,2,3,7,8-PeCDF and 1,2,3,6,7,8-/1,2,3,4,7,8-HCDF respectively (no. of determination shown in brackets). Although the Scatchard plots with 2,3,7,8-TCDD frequently were curvilinear at low concentrations of the radioligand, the plots obtained with the three PCDF radioligands were all linear. In addition, the Hill plot analysis of the data for all four radioligands gave slopes which were equal to one and the results are consistent with noncooperative ligand-receptor binding.

The influences of in vivo pretreatment with phenobarbitone (PB), 3-methylcholanthrene (3-MC), 2,2',4,4',5,5'-hexachlorobiphenyl (HCBP), and 3,3',4,4'-tetrachlorobiphenyl (TCBP) on cytocidal hepatotoxicity of two pyrrolizidine alkaloids, lasiocarpine (LC) and senecionine (SC), were compared in short-term primary cultures of rat hepatocytes. Toxicity was measured by release of lactate dehydrogenase (LDH) into culture medium at 24 h. LC was slightly more toxic to control hepatocytes than SC in the graded response range of 10-160 microM. PB and HCBP (a PB-type polychlorobiphenyl inducer) similarly potentiated toxicity of SC, and each diminished the degree to which cell killing by LC and SC was inhibited by SKF-525-A. By comparison, 3-MC and TCBP (a 3-MC-type PCB inducer) each diminished toxicity of SC but had little effect on toxicity of LC. Alpha-naphthoflavone (ANF) potentiated toxicity of both LC and SC in hepatocytes induced by 3-MC or TCBP but had little effect on responses of hepatocytes induced by either PB or HDBP. These results indicate that xenobiotics that induce similar patterns of cytochrome P-450 isozymes have qualitatively similar modulating influences on cytocidal hepatotoxicity of pyrrolizidine alkaloids in primary cultures. However, the observed modulating effects could not be explained solely on the basis of altered activation rates by the cytochrome P-450 species known to be induced by the various xenobiotics.

Holstein dairy cows weighing from 500 to 650 kg were given tritiated TCDD on crushed grain at a dosage level of either 75 or 0.05 μg/kg body weight, per os. The higher dosed animal was killed on day 7 while the 2 lower dosed animals were killed on day 14. In both the high and low dosed animals, radioactivity was eliminated mainly in the feces, but also in milk and urine. The major portion of the administered dose was eliminated via these routes in the first several days post-treatment. Tissue residues were found to be the highest in fat, followed by liver, kidney, muscle and brain.

The toxic Yusho oil contained a mixture of polychlorinated biphenyls (PCBs) which were contaminated by other halogenated aromatics including the highly toxic polychlorinated dibenzofurans (PCDFs). It has been reported that the PCBs and PCDFs which persist in the liver of victims still suffering from Yusho poisoning include the following compounds; 2,3′,4,4′,5-penta-,2,2′,4,4′,5,5′-,2,2′,3′,4,4′,5- and 2,3,3′,4,4′,5-hexa, 2,2′,3,4,4′,5,5′- and 2,2′,3,3′,4,4′,5-heptachlorobiphenyls and the 2,3,7,8-tetra-, 1,2,4,7,8-, 1,2,3,7,8- and 2,3,4,7,8-penta- and 1,2,3,4,7,8-hexachlorodibenzofurans. All of these PCBs and PCDFs have been synthesized and reconstituted to approximate their composition in human liver. A comparison of the dose-response effects of the reconstituted PCB and PCDF mixtures in causing weight loss, thymic atrophy and the induction of cytochrome P-448-dependent monooxygenases indicated that the PCDF mixture was at least 700 times more active than the PCBs. Since the ratio of PCBs/PCDFs persisting in Yusho patients' blood and liver was less than 600:1 and 5:1 respectively, the results suggest that the PCDFs are the major etiologic agent in Yusho poisoning.

Treatment of male Long Evans rats with doses of MCDF up to 200 μmol/kg did not significantly induce hepatic microsomal aryl hydrocarbon hydroxylase (AHH) or ethoxyresorufin O-deethylase (EROD) activities, whereas administration of 2,3,7,8-TCDD (16 nmol/kg) caused up to a 10 to 40-fold induction of these enzymes. Cotreatment of the rats with 2,3,7,8-TCDD (16 nmol/kg) and MCDF (50 μmol/kg) resulted in the inhibition of the monooxygenase enzyme induction response over a 72 hour period. Treatment of the animals with 2,3,7,8-TCDD alone caused an initial decrease in the concentration of the cytosolic Ah receptor followed by an increase of these levels (∼2 times higher than in control rats) after 72 hours. In contrast, MCDF treatment did not alter hepatic cytosolic Ah receptor levels and in the cotreatment studies (MCDF + TCDD), the effects of 2,3,7,8-TCDD on receptor levels was inhibited. Using [ 3 H]-2,3,7,8-TCDD (+ MCDF), the time-course accumulation of nuclear [ 3 H]-2,3,7,8-TCDD-receptor complexes was also investigated. Surprisingly, MCDF did not decrease occupied nuclear 2,3,7,8-TCDD-Ah receptor levels.

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.

The ED50s for the dose-response induction of hepatic microsomal aryl hydrocarbon hydroxylase (AHH) and ethoxyresorufin O-deethylase (EROD) by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in the female Sprague-Dawley rat were 3.3 and 2.7 nmol/kg, respectively. In contrast, the corresponding ED50 values for induction by the nontoxic 6-methyl-1,3,8-trichlorodibenzofuran (MCDF) were 524 and 578 mumol/kg for AHH and EROD, respectively, and TCDD was greater than 1.5 X 10(5) more potent than MCDF as an agonist for this response. Cotreatment of the female rats with MCDF (20, 50, or 100 mumol/kg) and TCDD (6.4 nmol/kg) showed that MCDF partially antagonized the induction of AHH and EROD by TCDD and this corresponded with results previously reported in the male rat. Like TCDD, MCDF also caused a dose-response decrease in uterine and hepatic cytosolic and nuclear estrogen (ERc and ERn) and progesterone (PRc and PRn) receptor levels. The relative TCDD/MCDF potencies for the reduction of uterine ERc, ERn, PRc, and PRn levels were 293, 569, 560, and 459, respectively, and comparable potency ratios (693, 409, 405, and 424, respectively) were observed in the liver. Since MCDF was active as an antiestrogen at dose levels which caused only minimal induction of hepatic monooxygenases, it is unlikely that induction of these enzymes and the subsequent increased metabolism of estradiol play a role in the antiestrogenic effects of MCDF (or TCDD). The reasons for the differences in the relative potency of MCDF for the "traditional" Ah receptor-mediated response (i.e., AHH induction) and the modulation of steroid hormone receptor binding levels are unknown. MCDF, a compound which exhibits relatively high TCDD receptor binding activity and low toxicity, represents a new class of nontoxic halogenated aromatic antiestrogens that can be utilized to further probe the mechanism of this response in model systems.

The relative activities of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and progesterone were determined in the 25 day old female Long Evans rats. Both 2,3,7,8-TCDD and progesterone treatment result in significant decreases in nuclear and cytosolic progesterone and estrogen receptor (PR and ER). The major difference between the two compounds is associated with the relative persistence of the effects of 2,3,7,8-TCDD. Using an in vitro uterine strip assay, similar effects on ER and PR levels were observed following 2,3,7,8-TCDD treatment on the uteri. Protein and RNA synthesis inhibitors, namely puromycin, cycloheximide and actinomycin D were preincubated with the uteri prior to progesterone or 2,3,7,8-TCDD treatment. Previous studies by Leavitt and coworkers suggested that progesterone-induced decreases in nuclear ER levels were dependent on both RNA and protein synthesis. With 2,3,7,8-TCDD, the protein synthesis inhibitors did not prevent the observed decreases in ER and PR levels, whereas the RNA synthesis inhibitor actinomycin D inhibited the effects of 2,3,7,8-TCDD on uterine ER and PR levels. These results suggest that the mechanism of 2,3,7,8-TCDD-mediated decreases in ER and PR levels is different from that of progesterone.

6-Methyl-1,3,8-trichlorodibenzofuran (MCDF) have been shown to inhibit TCDD-induced enzymeinduction both in rat and in cell culture. MCDF, alone or together with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), was administered to rats in order to investigate the ability of MCDF to reduce TCDD-induced vitamin A depletion. High doses of MCDF induced vitamin A-depletion, but no inhibitory effect on TCDD-induced depletion was seen in this experiment.

A thyroglobulin conjugate of dioxin (thyroglobulin-2 adipamide, 3,7,8-trichlorodibenzo-p-dioxin) (TG-TCDD) was used to immunize BALB/c mice. Hybridomas were produced by cell fusion between immune spleen cells and mouse myelomas SP2/0, P3, or NS1. In order to screen the thousands of resultant cultures for production of MoAb, a rapid, solid phase radioimmunoassay for antibody to dioxins was developed. This involved attaching bovine serum albumin coupled with trichlorodibenzo-p-dioxin (BSA-TCDD) to polystyrene plates to be used as a solid phase target antigen for reaction with MoAb. Fourteen hybridomas were identified that produced MoAb reacting with BSA-TCDD but not with BSA alone. Antibodies were tested for binding to BSA-aniline to eliminate those with limited binding specificity. Initial studies indicated that most MoAbs bound BSA-aniline as well as BSA-TCDD. More detailed analyses indicated that while most MoAbs showed some reaction with BSA-aniline, two showed preferential binding to BSA-TCDD of more than 200 fold whereas rabbit antisera demonstrated only a 5-fold discrimination. MoAb 391-1B was purified from mouse ascites fluid and after radioiodination, was tested for direct binding to BSA-TCDD or BSA-aniline. 125I MoAb showed no significant binding to BSA-aniline while demonstrating high binding to BSA-TCDD ( Ka = 4.5 × 1081 mole ). Experiments are in progress to use these MoAbs to develop sensitive radioimmunoassays or enzyme linked assays specific for 2,3,7,8-TCDD.

Several studies have reported that 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) acts as an antiestrogen in rodents and human breast cancer MCF-7 cells in culture. It has been proposed that these effects may be related to the induction of estradiol-2-hydroxylase activity which effects a decrease in cellular estradiol levels due to increased metabolism. This study reports the comparative antiestrogenic effects of TCDD and 6-methyl-1,3,8-trichlorodibenzofuran (MCDF) on cytosolic and nuclear estrogen and progesterone receptor levels in both the rat uterus and liver. MCDF, like TCDD, acted as an antiestrogen in the female rat, causing a dose-response decrease in uterine and hepatic cytosolic and nuclear estrogen and progesterone receptor levels; both compounds also antagonized the estradiolinduced increase in uterine wet weights. TCDD was 300 to 700 times more potent than MCDF as an antiestrogen, however the TCDD/MCDF potency ratios as inducers of hepatic monooxygenases were > 150,000. Moreover, the antiestrogenic effects of MCDF were observed at doses which caused minimal induction of hepatic monooxygenases. These results suggest that the antiestrogenicity of TCDD and related compounds is not due to increased metabolism of estradiol.

Administration of 2,3,7,8-TCDD (20 μg/kg) to female C57BL/6 mice gave >60% cleft palate in the litters. Previous studies have shown that cotreatment of the pregnant mice with 2,3,7,8-TCDD (20 μg/kg) and Aroclor 1254 (750 μmol/kg) resulted in complete protection of the animals from cleft palate. In contrast, a lower dose of Aroclor 1254 (250 μmol/kg) was a less effective 2,3,7,8-TCDD antagonist. Several PCB congeners, including 2,2′,5,5′-tetrachlorobiphenyl and 2,2′,4,4′,5,5′-hexachlorobiphenyl, which exhibit some Ah receptor binding activity were investigated as 2,3,7,8-TCDD antagonists in the in vivo teratogenicity assay system. Both PCB congeners significantly protected the mice from 2,3,7,8-TCDD-mediated cleft palate. The significance of these results and their mechanistic implications will be discussed.

The competitive receptor binding affinities of 13 2-substituted 3,7,8-trichlorodibenzo-p-dioxin (TrCDD) for the rat hepatic cytosolic 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) receptor protein was determined using [3H]-2,3,7,8-TCDD as the radioligand. The 2-CF3 substituted analog was the most active compound in this series (pEC50=8.50) and exhibited a higher binding affinity for the receptor than 2,3,7,8-TCDD. Multiple parameters linear regression analysis of the data gave Equation 1 which correlates binding affinities with the physiochemical characteristics of the substituents. In this equation π, ΔES and HB represent pEC50 (rat) = 7.196 + 0.600π − 0.255δEs + 1.683 HB (1) (0.140) (0.231) (0.181) (0.267) n = 11 s = 0.099 r = 0.998 substituent lipophilicity, van der Waals volume and hydrogen bonding capacity respectively, n is the number of substituents, s is the standard deviation and r is the correlation coefficient. The effects of substituents on the biologic (aryl hydrocarbon hydroxylase induction) and toxic (thymic atrophy and body weight loss) effects of these compounds was also determined in the immature male Wistar rat. The 2-halo substituted analogs (F, Cl, Br and I) were all highly toxic however the most active compound, 2-trifluoromethyl-3,7,8-trichlorodibenzo-p-dioxin, also exhibited the highest binding affinity for the receptor protein.

Holstein dairy cows weighing from 488 to 650 kg were given tritiated 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on crushed grain or Gerald silt loam soil as a single oral dose of 0.05 ug/kg body weight. The cows were killed 14 days posttreatment. In both the grain- and soil-treated animals, tissue residues were highest in the fat, followed by liver, kidney, muscle and brain. Secretion of radioactivity into milk was highest during the first 3–4 days posttreatment with only small quantities being secreted into the milk during the following 10 days. Hepatic aryl hydrocarbon hydroxylase and ethoxyresorufin O-deethylase appeared to be slightly elevated 14 days after treatment with either grain or soil-borne TCDD. The aryl hydrocarbon (Ah) receptor was not detected using the hydroxylapatite assay procedure.

Administration of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) to 21 day old female Long Evans rats resulted in a dose-dependent decrease in uterine and hepatic estrogen receptor (ER) levels. At a dose level of 80 ug/kg of 2,3,7,8-TCDD, uterine and hepatic ER levels were 54 and 41% of values observed in untreated animals. 2,3,7,8-TCDD also antagonized the estrogenicity of estradiol in the 21 day old rat. For example, administration of estradiol (15 ug/kg) resulted in a 194 and 155% increase in cytosolic and uterine ER levels and a 42% increase in uterine wet weight 48 hours after treatment. Pretreatment of the rats with 2,3,7,8-TCDD (80 ug/kg) prior to administration of estradiol (15 ug/kg) completely blocked the estrogenic effects of estradiol. In fact after cotreatment the uterine and hepatic ER levels were 51 and 54% lower than observed in the controls and the uterine wet weight was 20% lower than in the control animals. The comparative effects of 2,3,7,8-TCDD, 1,2,3,7,8-pentachlorodibenzo-p-dioxin (PeCDD), 1,3,7,8-TCDD and 1,2,4,7,8-PeCDD on the down regulation of uterine and hepatic ER levels were structure-dependent. There was an excellent correlation between the binding affinities of these congeners for the Ah receptor and their activity as antiestrogens and the results support a role for the Ah receptor in the down regulation of uterine and hepatic ER levels in the rat. (Supported by the Texas Agricultural Experiment Station.)

The photoinduced formation of the covalently labeled cytosolic and nuclear aryl hydrocarbon (Ah) receptors was studied using 2,3,7,8-[3H]tetrachlorodibenzo-p-dioxin (TCDD) as the photoaffinity label. Irradiation of TCDD alone at wavelengths of greater than 300 nm resulted in rapid degradation of this compound (t 1/2 = 8 min). In a separate experiment, the unliganded cytosolic Ah receptor was only slowly inactivated (t 1/2 = 48 min) using the greater than 300 nm light source. Preliminary experiments with rat hepatic cytosol did not result in significant formation of specifically bound [3H]TCDD-protein covalent adducts which could be visualized by autoradiography. Irradiation of [3H]TCDD-nuclear Ah receptor complexes isolated from mouse Hepa 1c1c7 cells for 15 min gave approximately a 40% overall yield of the radiolabeled Ah receptor protein adduct. Denaturing sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the [3H]TCDD-nuclear Ah receptor photoadduct gave a single major radiolabeled protein with an apparent molecular size of 91 kDa. The chromatographic properties of the control (dark) and photolabeled nuclear Ah receptor complexes were comparable using Sephacryl S-300 and DNA-Sepharose columns. Velocity sedimentation of both the control (dark) and irradiated nuclear Ah receptor complexes gave specifically bound peaks which sedimented at 6.5 S. However, the trichloroacetic acid-precipitable (buffer-reconstituted) [3H]TCDD-nuclear Ah receptor photo-covalent adduct was eluted from the Sephacryl S-300 column in the void volume and did not exhibit a specifically bound peak after velocity sedimentation analysis due to protein aggregate formation. In contrast, the elution profile of the aggregate on a DNA-Sepharose column was similar to that observed for the control (dark) and photolabeled complexes, which were eluted from the column with salt concentrations between 0.24 and 0.28 M. These photolabeling studies show that [3H] TCDD can act as a photoaffinity label for the Ah receptor and can be utilized as photoligand to probe further the structure and function of this protein.

The effects of treating rats with various pregnenolone-16 alpha-carbonitrile (PCN)-type inducers of cytochrome P-450p on the liver microsomal metabolism of aflatoxin B1 (AFB1) were investigated. Treatment of male rats with PCN resulted in a 6-fold increase in the 9-hydroxylation of AFB1 to aflatoxin Q1 (AFQ1). Treatment of female rats with PCN resulted in a 16-fold increase in the formation of AFQ1. The age-dependent decline in constitutive cytochrome P-450p levels in female but not male rats resulted in a sex difference in the formation of AFQ1 in liver microsomes from untreated rats (male:female approximately 3:1). The formation of AFQ1 was stimulated up to 5.4-fold when liver microsomes from triacetyloleandomycin (TAO)-treated rats were treated with potassium ferricyanide, which dissociates the complex between cytochrome P-450p and TAO. Treatment of male rats with the cytochrome P-450p inducer, dexamethasone, increased (approximately 7-fold) the 9-hydroxylation of AFB1 to AFQ1 by liver microsomes, and also enhanced (approximately 2-fold) the microsomal activation of AFB1 to metabolites that were mutagenic to Salmonella typhimurium TA98 and TA100. These results indicate that the 9-hydroxylation of AFB1 to AFQ1 is catalyzed by rat liver microsomal cytochrome P-450p.

The effects of the higher chlorinated benzene and phenol congeners as inducers of the hepatic microsomal drug-metabolizing enzymes have been determined in the immature male Wistar rat by comparing the enzymic, electrophoretic, and spectral properties of the microsomes. 3,4,5-Trichiorophenol, 1,2,4,5-tetrachlorobenzene, 1,2,3,5-tetrachlorobenzene, 1,2,4-trichlorobenzene, and pentachlorobenzene induced 4-dimethylaminoantipyrine (DMAP) N-demethylase, an enzyme induced by phenobarbitone (PB) and several PB-type inducers. Hexachlorobenzene induced DMAP N-demethylase and aldrin epoxidase, two PB-inducible enzymes, and benzo[a]pyrene hydroxylase and ethoxyresorufin O-deethylase, two enzymes induced by 3-methylcholanthrene (MC). This mixed-type induction pattern has been previously reported for hexachlorobenzene. The remaining higher chlorinated benzene and phenol congeners were inactive as inducers of the drug-metabolizing enzymes in the immature male Wistar rats.

The mass spectrometric fragmentations of fifteen steryl methyl ethers have been recorded. In all cases strong molecular ions were obtained as well as significant variation in the spectra according to the stereochemistry of the A/B ring junction, the presence or absence of Δ5 unsaturation, and the position of the unsaturation in the side chain. The highly characteristic mass spectral data coupled with the excellent gas chromatographic properties of steryl methyl ethers indicate their potential value in gas chromatographicmass spectrometric structure analysis.

2-Hydroxy-3,7,8-trichlorodibenzo-p-dioxin has been identified as a major urinary metabolite of 2,3,7,8-TCDD in several laboratory animal studies. This compound was synthesized via coupling of 2,5-dichloro-4-nitroanisole with 4,5-dichlorocatechol in HMPA. The resultant 3-methoxy-3,7,8-trichlorodibenzo-p-dioxin product was demethylated in boron tribromide/methylene chloride to give 2-hydroxy-3,7,8-trichlorodibenzo-p-dioxin. The metabolite was administered at dose levels of 100, 1000 and 5000 ug/kg to immature male Wistar rats and the effects of this compound on body weight loss, thymic atrophy and hepatic microsomal monooxygenase enzyme induction were determined. Comparable dose-response studies for 2,3,7,8-TCDD were also carried out. At dose levels of 4–8 ug/kg 2,3,7,8-TCDD caused significant body weight loss and thymic atrophy in the Wistar rats whereas 2-hydroxy-3,7,8-trichlorodibenzo-p-dioxin did not elicit these toxic effects at a dose level of 5000 ug/kg. The ED 50 values for the induction of hepatic microsomal benzo[a]pyrene hydroxylase and ethoxyresorufin O-deethylase were 1.25 and 2.35 ug/kg for 2,3,7,8-TCDD and 2,820 and 3550 ug/kg for 2-hydroxy-3,7,8-trichlorodibenzo-p-dioxin respectively. These results demonstrated that 2-hydroxy-3,7,8-trichlorodibenzo-p-dioxin is at least 3 orders of magnitude less toxic than 2,3,7,8-TCDD and this is consistent with the lower receptor binding affinity of the metabolite coupled with the expected more rapid clearance of this compound.

Previous studies have demonstrated that the in vitro aryl hydrocarbon hydroxylase (AHH) and cytosolic receptor binding bioassays can be utilized to quantitatively estimate the potential in vivo toxicities of polychlorinated dibenzo-p-dioxins (PCDDs), dibenzofurans (PCDFs) and their mixtures (from municipal fly ash extracts). This study reports the use of the two bi⊙assays in estimating the levels of 3,3′,4,4′-tetrachloroazobenzene in several commercial products which contain or are derived from 3,4-dichloroaniline. The AHH induction and receptor binding EC 50 values for 3,3′,4,4′-tetrachloroazobenzene were 5.60 × 10 −7 and 5.60 × 10 −6 M respectively. The levels of 3,3′,4,4′-tetrachloroazobenzene (TCAB) in commercial samples of Diuron, Linuron and 3,4-dichloroaniline were determined by gas chromatographic (GC) analysis and the concentrations varied from trace levels to 2200 ug/g. Preliminary bioassays of the commercial compounds and crude extracts (BC) of these products gave contradictory results which did not correlate with the GC analytical data. Presumably there are impurities or coextractives which interfere with the sensitivity of the bioassays. The crude extracts were purified by column chromatography and were assayed using both bioassays. For most of the cleaned up extracts there was an excellent correlation between the concentrations of 3,3′,4,4′-TCAB determined by the GC and receptor binding procedures.

Treatment of immature male C57BL/6J mice with 2,2′,4,4′,5,5′-hexachlorobiphenyl (HCB, 100–1000 umol/kg) resulted in a 44–200% increase in levels of the 2,3,7,8-TCDD hepatic cytosolic receptor protein for up to 14 days. The increased binding was not due to an alteration of the affinity of 2,3,7,8-TCDD for the receptor as shown by Scatchard analysis. In contrast treatment of immature DBA/2 mice with 2,2′,4,4′,5,5′-HCB did not result in increased hepatic cytosolic receptor levels which were non-detectable in the treated and untreated animals. C57BL/6 mice were pretreated with 2,2′,4,4′,5,5′-HCB (500 umol/kg) and after 7 days 2,3,7,8-TCDD (0.32 ug/kg) was administered by i.p. injection; 14 days later the hepatic microsomal aryl hydrocarbon hydroxylase (AHH) and ethoxyresorufin O-deethylase (EROD) activities were 587 and 1383 pmol product/mg protein/min respectively. In contrast, these activities were 340 and 437 pmol/mg protein/min in animals treated with only 2,3,7,8-TCDD. These synergistic effects were only seen in mice treated with submaximal enzymeinducing doses of 2,3,7,8-TCDD. Treatment of DBA/2 mice with 2,2′,4,4′,5,5′-HCB (500 umol/kg) and varying dose levels of 2,3,7,8-TCDD (10–5000 nmol/kg) resulted in apparent synergistic AHH and EROD induction activities in mice treated with submaximal and maximal enzyme inducing doses of 2,3,7,8-TCDD.