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Nine catfish fillets, three catfish nuggets, two feed samples, and one pond sediment were analyzed for PCDD, PCDF, and PCB. Farm-raised catfish from Mississippi, Alabama, and Arkansas contained significant levels of 2,3,7,8-substituted PCDD and PCDF. In addition, a large number of non-2,3,7,8-substituted congeners were present in all samples. The catfish fillets and catfish nuggets also contained high concentrations of dioxin-like PCB, as well as a number of non-dioxin-like PCB. The TEQ based on PCDD and PCDF ranged from 9.5 to 43.0 pg/g lipid and the TEQ based on PCB ranged from 0.45 to 4.9 pg/g lipid for all catfish samples. The dioxin-like PCB contributed 4-16% to the total TEQ (PCDD/PCDF/PCB) for the catfish samples. The major source for the PCDD, PCDF, and PCB appears to be from feed and not from pond sediment. Immunoreactive CYP1A protein was elevated 2.5 fold in the pond-raised catfish compared to the aquarium-raised one. The results of this study suggest that the PCDD/PCDF are more important than the PCB in the CYP1A induction.

Hazard and risk assessment of individual toxic chemicals utilizes data obtained from chronic toxicity and carcinogenicity studies in laboratory animals for regulating emission, cleanup or intake levels for individual chemicals. This approach can be utilized for problems associated with a single chemical emitted from a point source; however, in most situations, toxic chemicals are formed and emitted into the environment as complex mixtures of different structural classes of toxic/carcinogenic chemicals. Methodologies for risk assessment of complex mixtures have been developed for polychlorinated dibenzo‐p‐dioxins (PCDDs) and dibenzofurans (PCDFs) which are found as industrial and combustion by‐products. This methodology is based on the common mechanism of action for these chemicals and utilizes the most toxic PCDD/PCDF congener, 2, 3,7,8‐tetrachlorodibenzo‐p‐dioxin (TCDD), as a reference compound. The relative potencies or toxic equivalency factors (TEFs) are assigned to the other relevant PCDD/PCDF congen...

In order to compare a standard chemical-based risk assessment with in vitro genotoxicity assays, two complex environmental mixtures from a wood-preserving site were analyzed in the Salmonella/microsome and E. coli prophage induction assays. Using GC/MS, sample 003 was found to contain relatively low levels of polycyclic aromatic hydrocarbons (PAHs) and elevated levels of polychlorinated dibenzo-p-dioxins (PCDDs), while sample 005 had higher levels of PAHs and relatively low levels of PCDDs. The complex mixtures were sequentially extracted with methylene chloride and methanol for analysis in Salmonella, or extracted with a 1:1 hexane:acetone mixture for analysis in the E. coli prophage induction assay. At a dose of 1.0 mg/plate in Salmonella strain TA98 with metabolic activation, the methanol extract of sample 003 induced 197 net revertants, while sample 005 induced 436 net revertants. In the prophage induction assay, with activation, the hexane:acetone extract of sample 003 induced a genotoxic response that was slightly lower than that observed with sample 005. The estimated incremental carcinogenic risk for ingestion of PAHs was 1.5E – 3 for sample 003, while for sample 005 the estimated risk was 1.5E – 2. Thus, the sample which induced the maximum response in both bioassays also had the highest estimated cancer risk. However, the frequency of PAH-DNA adducts in both skin and liver tissues was appreciably higher with sample 003 than with sample 005. A combined testing protocol, using both biological and chemical analysis, therefore provides more accurate information from which to assess risk than the use of either method alone.

The effects of 1 nM 17β-estradiol, 1 nM 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and their combination on the formation of intracellular cytosolic 52- and 34-kDa proteins was determined using a commercially available cathepsin D radioimmunoassay procedure (ELSA cath-D™ kit) which recognizes both the 34- and 52-kDa proteins. In control (ethanol-treated) cells, the 52-kDa protein levels were 305 ± 49 fmol/mg protein (100%) whereas in cells treated with 1 nM 17β-estradiol, 1 nM TCDD or their combination the levels were 1223 ± 142 (401%), 478 ± 54 (150%) and 227 ± 33 (74%). These results confirm that TCDD inhibits the 17β-estradiol-induced intracellular formation of the 52-/34-kDa proteins and further extends the antiestrogenic properties of this compound.

Using [3H]2,3,7,8-tetrachlorodibenzo-p-dioxin ([3H]TCDD) or 7-[125I]-iodo-2,3-dibromodibenzo-p-dioxin ([125I]DBDD) as high affinity photoaffinity ligands, the photoaffinity-labeled nuclear Ah receptor complexes from human cell lines have been analyzed by denaturing sodium dodecyl sulfate polyacrylamide gel electrophoresis. The results show that the apparent molecular mass for the Ah receptor from several of these cell lines was 110-kDa and no significant variations were observed. However, there were significant differences in the apparent molecular weights of the nuclear Ah receptor complexes. For example, in the estrogen receptor (ER)-positive (MCF-7) and ER-negative (MDA-MB-231) human breast cancer cell lines, the apparent molecular weights of the nuclear receptor complexes were 171,000 and 192,000, respectively. These results indicate that there are differences in the molecular weights of the human nuclear Ah receptor complexes and the intraspecies heterogeneity may be associated with the non-ligand binding protein and not the Ah receptor.

Recent studies have reported that, like 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), interleukin-1α inhibits 17β-estradiol-induced cell proliferation and [3H]thymidine uptake and also causes a downregulation of the nuclear estrogen receptor (ER)-positive MCF-7 human breast cancer cells. These results suggest a possible relationship between TCDD and interleukin-1α in human breast cancer cells. After treatment of MCF-7 cells with 1 nM TCDD, the media was changed every 48 hours over a period of 7 days and used to support the growth of cells which were not treated with TCDD. The results showed that media from cells treated with TCDD did not inhibit the growth of control or 17β-estradiol-treated MCF-7 cells. These results suggested that MCF-7 cells exposed to TCDD did not secrete anti-growth factors into the media. In a separate study, the media from control cells and those treated with 1 nM 17β-estradiol, 1 nM TCDD and 17β-estradiol plus TCDD were analyzed for interleukin-1α using commercially-available antibodies. The results showed that interleukin-1α was not detectable in media from any of the treatment groups and indicate that although interleukin-1α and TCDD cause several common responses in ER-positive human breast cancer cell lines, their modes of action are independent.

Langdon and coworkers (1990) have characterized a series of estrogen receptor (ER)-positive and ER-negative human ovarian carcinoma cell lines. Previous studies have demonstrated that TCDD exhibits a broad spectrum of antiestrogenic activities in ER-positive human breast cancer cell lines. Studies in this laboratory have shown that after treatment with 10 nM [3H]TCDD, specifically-bound aryl hydrocarbon (Ah) receptor complexes were identified in the cytosolic and nuclear fractions of the ER-positive PE01, PE04 and PE06 human ovarian carcinoma cell lines. Preliminary studies indicate that the molecular properties of the nuclear and cytosolic Ah receptor complexes in the ovarian carcinoma cells were similar to those reported for the Ah receptor from other human tissues and cells. The effects of TCDD on cytochrome P-4501A1 and the growth of these cell lines will be discussed.

Exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) alters testicular function; however, its effect on daily sperm production and Leydig cell volume has not been determined in adult rats. Adult male Sprague-Dawley rats (300–350 g) received 0, 12.5, 25.0, or 50.0 μg/kg body weight doses of TCDD by a single intraperitoneal injection. The body weight was reduced (P 0.05) to have reduced Leydig cell volume after TCDD treatment, and rats in Exp. 2 revealed a dose-dependent reduction (P

The effects of polychlorinated dibenzofurans (PCDFs) on hepatic I-compound DNA adducts in female Sprague-Dawley rats were determined at 2 dose levels. In the first experiments, rats (4 per group) were treated with each of 3 polychlorinated dibenzofuran (PCDF) congeners at 20 μg/kg/week or 2,3,7,8-tetrachlorodibenzo- p -dioxin (TCDD) at 10 μg/kg/week for 4 weeks. Effects on total I-compound levels when compared with age-matched control animals (corn oil) were as follows: control (116) > 1,2,4,7,8-pentachlorodibenzofuran (PeCDF) (112) > 1,2,3,7,8-PeCDF (93) > 2,3,4,7,8-PeCDF (91) > TCDD (72) where the numbers in parentheses were the number of I-compounds in 10 9 DNA nucleotides. In the second experiment, rats (5 per group) were treated with each of the congeners at a higher dose of 100 μg/kg/week for 4 weeks. The effects were as follows: control (123) ≈ 1,2,4,7,8-PeCDF (125) > 1,2,3,7,8-PeCDF (98) > 2,3,4,7,8-PeCDF (75) ≈ 2,3,4,6,7,8-hexachlorodibenzofuran (HCDF) (75). The active compounds (1,2,3,7,8-PeCDF, 2,3,4,7,8-PeCDF and 2,3,4,6,7,8-HCDF) were those that exhibited high cytosolic Ah receptor binding affinities (1.5 – 7.5 × 10 −8 M) and were also potent AHH inducers. The inactive compound (1,2,4,7,8-PeCDF) was less active in the receptor binding and enzyme induction assays. The results showed that there was a structure-dependent correlation between the Ah receptor agonist activity of a congener and its potency to reduce I-compounds in female Sprague-Dawley rat liver.

2,3,7,8-Tetrachlorodibenzo- p -dioxin (TCDD) elicits a broad spectrum of antiestrogenic responses in MCF-7 human breast cancer cells. Treatment of the cells with insulin (0.001 – 10.0 μg/ml) caused a significant concentration-dependent increase in cell proliferation at concentrations of 0.01 – 10.0 μg/ml with a maximum response observed at 1.0 μg/ml. Treatment of the cells with TCDD (0.01 to 10 nM) caused some decrease in cell growth at the high concentrations. Cotreatment of the cells with insulin plus TCDD at several different concentrations showed that TCDD at a concentration as low as 0.1 nM inhibited insulin-induced cell proliferation. In a parallel study, insulin also caused a concentration-dependent increase in [ 3 H]thymidine uptake into cellular DNA at concentrations of 0.01, 0.1 and 1.0 μg/ml and TCDD (0.1 nM) significantly inhibited this response at all 3 concentrations of insulin. These results illustrate the interaction between TCDD and insulin in MCF-7 cells and further extend the antimitogenic activity of TCDD in human breast cancer cell lines.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) exhibits a broad spectrum of antiestrogenic effects in estrogen receptor (ER) positive MCF-7 human breast cancer cells. The proliferation of MCF-7 cells in minimal media can also be stimulated by other mitogens, including insulin, insulin-like growth factor-1 (IGF-1), transforming growth factor α (TGF-α) and epidermal growth factor (EGF). The interactions of TCDD and these mitogens showed that TCDD inhibited growth factor-induced cell proliferation and the [3H]-thymidine uptake in the MCF-7 cells. A more detailed analysis of the interaction of TCDD and IGF-1 showed that treatment of the cells with IGF-1 (or 17s-estradiol) caused a 100% increase in the number of membrane-bound IGF-1 receptor binding sites whereas cotreatment with TCDD completely blocked these induced effects. In contrast, the K d values for the binding of [125I]-IGF-1 with its receptor were not significantly different in cells from all the treatment groups. Parallel studies in T47-D cells showed that there was differential-responsiveness to the proliferative effects of these mitogens; however, TCDD also exhibited comparable antimitogenic activities in these cell lines.

Bioremediation is gaining acceptance as a viable alternative for remediating contaminated soils at Superfund sites. However, it has the potential for resulting in the production of intermediate breakdown products which may be more toxic and soluble than the parent compound. This study was designed to monitor the mutagenic activity of contaminated soils from an abandoned wood preserving facility undergoing bioremediation in an intensively managed land treatment unit (LTU). Sample collection included two waste samples, twelve soil samples from the LTU, one on- site control soil sample, and one off-site control sample. Samples were sequentially extracted with methylene chloride and methanol, and the residues from these extractions were dissolved in dimethyl sulfoxide for testing with the Salmonella/microsome assay (Ames test). Each extract was tested using strain TA98 with and without metabolic activation, and the results were used to measure the bacterial mutagenicity which is an indicator of the carcinogenic potential. Although chemical analysis had suggested that biodegradation had reduced the concentration of toxic organics to below method detection limits, the results of bioassay testing detected mutagenic chemicals still present in both lifts of soil in the LTU. The weighted activity (revertants per gram of soil) of the soil from the upper lift which had been in the LTU for only five weeks exceeded two times the background, whereas the weighted activities of soil samples from the lower lift which had been in the LTU for three months were within background values for this site. These data indicate that three months of remediation in an intensively managed LTU is sufficient to reduce the weighted activity of contaminated soil from this facility to near background levels.

A series of nitropolychlorodibenzo- p -dioxins (NPCDDs) was found to be stable to base in the dark with one exception, and only slowly destroyed by ultraviolet light λ > 300 nm in the absence of base. However, they photolyzed rapidly in the presence of dilute sodium hydroxide. The reactions were followed using an in vitro bioassay based on the affinity of the substrates for the murine hepatic Ah receptor; this allows the reactions to be followed in extremely dilute solution (μM or less). A possible methodology for the destruction of polychlorinated dibenzo- p -dioxins involves nitration followed by photolysis in base.

Chlorinated trans stilbenes bind with high affinity to the cytosolic Ah receptor from Wistar rat liver. The EC50 values for competition with [3H]‐2,3,7,8‐tetrachlorodibenzo‐p‐dioxin (TCDD) cover a range from 10–400 nM, but with no apparent relationship between molecular structure and binding activity. These compounds induce monooxygenase enzyme activity in rat hepatoma H‐4‐II E cells in culture only weakly, again with no apparent dependence on molecular structure, and the weakest enzyme inducers act as antagonists for the induction of these enzymes by TCDD. In vivo administration of 3,3’,4,4'‐tetrachloro‐tans‐stilbene in the rat leads to decreased Ah receptor levels.

The competitive binding affinities of thirteen 7-substituted-2,3,-dichlorodibenzo-p-dioxins to the human Ah placental cytosolic Ah receptor were determined versus [ 3 H ]-2,3,7,8- tetrachlorodibenzo -p- dioxin (TCDD) as the radioligand. Multiple parameter linear regression analysis of the competitive binding EC50 values for these compounds gave the following equation: pEC 50 (M) =6.246+1.632 π − 1.764σ m° + 1.282 HB were π is the substituent lipophilicity, σm° the meta-directing electronegativity and HB the hydrogen binding capacity. The equation obtained using human placental receptor was different than correlations previously derived for the binding of the same series of compounds to the rat, mouse, guinea pig or hamster cytosolic Ah receptor, providing further evidence for interspecies differences in the properties of the Ah receptor protein.

The concentration-dependent induction of aryl hydrocarbon hydroxylase (AHH) and ethoxyresorufin-O-deethylase (EROD) activities by several PCB congeners was determined in primary chick embryo hepatocytes in culture. The most active inducers were the coplanar PCBs, 3,3′,4,4′-tetra- and 3,3′,4,4′5-pentachlorobiphenyl (pentaCB) with EC 50 values of 1.2 × 10 −9 and 1.5 × 10 −9 M (AHH induction) and 2.3 × 10 −9 and 1.2 × 10 −9 M (EROD induction) respectively. The corresponding EC 50 values for 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) were 2.4 × 10 −11 and 7.2 × 10 −11 M for AHH and EROD induction respectively. Three monoortho coplanar PCB analogs, 2,3,3′,4,4′-pentaCB, 2,3′,4,4′,5-pentaCB and 2,3,3′,4,4′,5-hexaCB also exhibited induction activity but their corresponding EC 50 values were much higher than observed for the coplanar PCBs. Several di- and triortho-substituted PCB congeners were weakly active as inducers of AHH and EROD activity. The structure-induction relationships for the coplanar and monoortho coplanar PCBs in the chick embryo hepatocytes were comparable to those previously reported in rat hepatoma H-4-II E cells and in in vivo studies.

Vapor pressures are important parameters for the modelling of the environmental fate and incineration behavior of halogenated dibenzo-dioxins and dibenzofurans (1). Correlation methods play an important role in the determination of thermal properties of polyhalogenated dioxins and furans (2,3) as there are hundreds of homologs and isomers whose synthesis in their pure form can be very difficult. Many of the compounds are highly toxic and their impact on the environment must be considered. The hazards associated with 2,3,7,8-T4CDD are well known. The measurement of thermal properties is involved for safety reasons, and the required quantities of these substances often fall within the milligram range. Correlation methods permit the estimation of the substance properties of hundreds of compounds from a limited set of measurements on the least toxic species. The correlation method (2,3) has in the past been used a) to predict enthalpies of fusion and boiling points from a set of vapor pressure results from polychloro dibenzo-dioxins, dibenzofurans and xanthenes; b) to test the self consistency of this set of measured vapor pressures; c) to predict the vapor pressures of solid 2,3,7,8-T4CDD and d) predict vapor pressures for twenty-nine halogenated dibenzo-dioxins and for fifty-five polychloro dibenzofurans (3). The correlation method was based on experimental vapor pressure data for one to four chlorinated congeners and on the octachloro dibenzo- p-dioxins and furans. We are presently extending the data set by including pentahexa- and heptachloro dibenzo-p-dioxins (1,2,3,4,7-P5CDD, 1,2,3,4,7,8-H6CDD, 1,2,3,4,6,7,8-H7CDD) and dibenzofurans (1,2,3,4,8-P5CDF, 1,2,4,7,8-P5CDF, 1,2,3,4,7,8-H6CDF, 1,2,4,6,7,8-H6CDF, 1,2,4,6,8,9-H6CDF, 1,2,3,4,6,7,8-H7CDF, 1,2,3,4,6,7,9-H7CDF, 1,2,3,4,6,8,9-H7CDF). The influence of exchanging chlorine against bromine has also been studied by recording vapor pressure curves on the following brominated dibenzo-p-dioxin congeners: 2,3,-D2BDD, 1,2,3,4-T4BDD and 1234678-H7BDF.

Treatment of adult female rats with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD, 1 μg and 5 μg/kg/week) over a period of 4 weeks resulted in a dose-dependent decrease in the levels of hepatic I-compounds (DNA adducts) as determined by the 32 P-postlabeling procedure. In adult male Sprague Dawley rats TCDD did not change the levels of total hepatic I-spots. The significance of these sex-specific effects and other data will be discussed.

Wild-type mouse Hepa 1c1c7 cells are responsive to the effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin and related compounds. This cell line and nonresponsive mutants (TAOc1BPrc1 and BPrc1) have been used to study properties of cytosolic (unactivated and activated) and nuclear Ah receptor-[3H]TCDD complexes as a model for investigating the molecular mechanism of action of halogenated aromatic hydrocarbons. Incubation of cytosolic [3H]TCDD-aryl hydrocarbon (Ah) complexes from wild-type Hepa 1c1c7 cell for 16h at 4° C in 0.4 M KCl resulted in the formation of transformed specifically-bound receptor complexes which exhibited increased binding affinity on DNA-Sepharose columns. The elution properties of the peak with the highest DNA binding affinity were similar to the elution profiles of the nuclear receptor complex isolated from wild-type cells. Comparable in vitro transformation and DNA binding properties were also observed using hepatic cytosol from C57BL/6J mice. TAOc1BPrc1 (class I) nonresponsive mutant cells were characterized by relatively low levels of the cytosolic and nuclear Ah receptor complexes. The cytosolic TCDD-Ah receptor complex did not undergo in vitro transformation. The BPrc1 (class II) variant cell lines contained levels of cytosolic receptor which were comparable to those observed in the wild type cells, but there were significantly reduced levels of nuclear receptor complex in the class II variant cell line. Incubation of the cytosolic receptor complex from these mutant cells at 4° C for 16 h or 20° C for 2 h did not result in transformation of the cytosolic receptor complex into forms which exhibit increased retention on DNA-Sepharose columns.

Several commercial PCBs, including Aroclors 1260, 1254, 1248, 1242 and 1232, were immunotoxic in C57BL/6 mice, however the results showed that the lower chlorinated biphenyls (Aroclor 1232 and 1242) were less toxic than the higher chlorinated mixtures. Treatment of C57BL/6J mice with an immunotoxic dose of 2,3,7,8-TCDD (3.7 nmol/kg) and a dose (25 mg/kg) of the commercial PCBs which was not immunotoxic showed that the PCB mixtures antagonized the 2,3,7,8-TCDD-mediated decrease in the plaque-forming cell response to sheep erythrocytes. The structure-dependent effects of individual PCB congeners to act as 2,3,7,8-TCDD antagonists were also investigated and only 2 compounds, namely 2,2′,4,4′,5,5′-and 2,3,3′,4,5,5′-hexachlorobiphenyl exhibited antagonist activity comparable to that observed for Aroclor 1254 as a 2,3,7,8-TCDD antagonist. The implications of these results will be discussed.

A series of [3H]-PCDD and [3H]-PCDF congeners of high specific activity were prepared by the chlorination of 1,6-3H2]-dibenzo-p-dioxin and 1,4,6-[3H3]-dibenzofuran followed by high pressure liquid chromatographic separation of the resultant mixtures. The compounds synthesized were tritiated 2,3,7,-TrCDD; 2,3,7,8-TCDD; 1,2,3,7,8-PeCDD; 1,2,7,8-TCDF; 2,3,7,8-TCDF; and 1,2,3,7,8-PeCDF. Direct binding studies with male Wistar rat hepatic cytosol showed that the KD values for all six congeners were approximately 5–10 nM. The lack of structure-dependent binding affinities to the Ah receptor is in contrast to previous competitive receptor binding studies and the well-known structure-induction and structure-toxicity relationships for these compounds. The kinetics of binding for each of these ligands to the Ah receptor, and the rate of thermal inactivation of the unliganded receptor were determined at several temperatures. Activation parameters were obtained using both graphical and computational methods; they varied with the identity of the ligand and exhibited substantial experimental uncertainties. The activation parameters were used to computationally determine revised KD's for the congeners which were in the pM range.

The dose-response effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), 2,3,7,8-tetrachlorodibenzofuran (TCDF) and 1,2,4,7,8-pentachlorodibenzo-p-dioxin (PeCDD) as inducers of aryl hydrocarbon hydroxylase (AHH) and ethoxyresorufin O-deethylase (EROD) were determined for 3 human breast cancer cell lines. The T47-D and MCF-7 cell lines were highly responsive to the induction effects of TCDD and TCDF. EC50 values were approximately 1 × 10−9 M for TCDD and TCDF in both cell lines and in both enzyme assays. These results, coupled with the low activity of the PeCDD congener were consistent with an Ah receptor mediated-induction process. The MDA-MB-231 cell line was not responsive to TCDD, TCDF or PeCDD. Preliminary data demonstrated that pretreatment of MCF-7 cells with TCDD resulted in a substantial decrease in the amount of nuclear estrogen receptor bound by [3H]-estradiol. Additional studies using estrogen receptor antibodies confirmed the reduction of the nuclear estrogen receptor protein in MCF-7 cells by TCDD.

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.

Twenty one (21) of the twenty eight (28) theoretically possible pentachlorodibenzofurans (p 5 CDFs) were synthesized and purified. Their retention indices were determined and were found to correlate well to those predicted using a previously developed mathematical model. Gas chromatographic/mass spectrometric (GC/MS) analysis of these compounds revealed that their electron-impact (EI) mass spectra were essentially identical with no significant variations due to positioning of the chlorines. Additional GC/MS experiments indicated that higher electron energies (70 eV as opposed to lower energies) were more suitable for analysis of these compounds due to higher molecular and fragment ion intensities.

Both ornithine decarboxylase (ODC) and aryl hydrocarbon hydroxylase (AHH) are inducible by 2,3,7,8-tetrachlorodibenzo-p-dioxin (2,3,7,8-TCDD) in rodents. Moreover, in genetically inbred mice the induction of both enzymes appears to be mediated through the ligand-activated Ah receptor protein. In rats, there is a 6 hour peak of induced ODC activity which occurs after administration of high (250 ug/kg) but not low (1–100 ug/kg) doses of 2,3,7,8-TCDD; the 6 hour peak of activity is not observed at any other times points following exposure to the toxin. The induction of AHH is a time and dose-dependent process in which enzyme activity is maximized 72 hours after treatment with 2,3,7,8-TCDD and persists for several days. Coadministration of -difluoromethylornithine (DFMO) and a toxic dose of 2,3,7,8-TCDD (250 umol/kg) completely eliminated the induction of ODC but did not effect the induction of AHH or alter the toxicity (thymic atrophy) of 2,3,7,8-TCDD. These results confirm that both ODC and AHH are induced by 2,3,7,8-TCDD and that the induction of the former enzyme does not play a role in modulating AHH induction or thymic atrophy.

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.

The Ah or 2,3,7,8-TCDD receptor protein plays an important role in mediating the biologic, toxic and genotoxic effects of aryl and halogenated aryl hydrocarbons. An assessment of the physicochemical parameters which facilitate ligand: receptor interactions was determined by multiple parameter linear regression analysis of the relative receptor binding affinities of a series of 7-substituted-2,3-dichlorodibenzo-p-dixoins (Eq 1), 8-substituted-2,3-di (Eq. 2) and 2,3,4-trichlorodibenzofurans (Eq 3). The results demonstrate that substituent lipophilicity (π) and molecular volumes were Eq. 1 log (I/EC 50 ) = 1.24 π + 6.10 Eq. 2 log (I/EC 50 ) = 1.10 π + 5.19 Eq. 3 log (I/EC 50 ) = 1.09 π + 5.77 the major determinant factors governing interactions between the rat hepatic cytosolic receptor protein and the diverse substituted ligands. It has been shown that there are marked differences in species sensitivity to 2,3,7,8-TCDD (i.e. guinea pig > rat ∼ maice > hamster) although hepatic receptor levels in these species are comparable. QSAR analysis of the receptor binding EC50 data for the 7-substituted-2,3-dichlorodibenzo-p-dioxins using rat (Eq. 1), mouse (Eq. 4), guinea pig (Eq. 5) and hamster (Eq. 6) hepatic cytosol demonstrated that there were significant differences in these equations. However it was also noted that Eq. 4 log (I/EC 50 ) = 0.95 + 0.93 E s + 5.28 Eq. 5 log (I/EC 50 ) = 0.94 + 0.579 σ p + 6.13 Eq. 6 log (I/EC 50 ) = 0.70 + 1.227 σ p + 6.38 physicochemical factors which are important for ligand-receptor interactions were identical for the most sensitive (guinea pig) and least sensitive (hamster) species. These results indicate that events which follow the initial ligand-receptor interaction must be the major factors which determine species sensitivity to 2,3,7,8-TCDD.

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 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.

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.

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.

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 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.

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.

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.