Hepatic Microsomal Aryl Hydrocarbon Hydroxylase Research Articles

Biomarkers of contaminant exposure in common terns and black‐crowned night herons in the Great Lakes

AbstractMorphological and biochemical indexes of contaminant exposure were examined in hatching common terns (Sterna hirundo) and black‐crowned night herons (Nycticorax nycticorax) from industrialized and nonindustrialized locations in the Great Lakes. In 1984, naturally incubated, pipping common tern and black‐crowned night heron embryos collected from industrialized locations exhibited smaller femur‐length‐to‐body‐weight ratios, elevated hepatic microsomal aryl hydrocarbon hydroxylase (AHH) activities, and lower hepatic DNA concentrations (P < 0.05). In addition, a high incidence of subcutaneous edema was noted in pipping herons (P < 0.01). In 1985, reduced hatching success was observed for laboratory‐incubated common tern eggs collected from the industrialized sites, compared to nonindustrialized sites (P < 0.01). Day‐old hatchlings exhibited reduced femur‐length‐to‐body‐weight ratio, developmental anomalies, and elevated hepatic AHH activity (P < 0.05). For hatching common terns studied in 1984 and 1985, femur‐length‐to‐body‐weight ratio was inversely related to AHH activity (r = −0.67, P < 0.05) and inversely related to log‐transformed PCB concentrations (r = −0.70, P ≤ 0.05) of unincubated eggs from the same colony. The activity of AHH in hatching terns was also directly related (r = 0.71, P ≤ 0.05) to log‐transformed PCB concentrations in unincubated eggs. Other examined contaminants, including DDE, other organochlorine pesticides, and mercury, were not directly related to these effects.

Comparative Potencies of Aroclors 1232, 1242, 1248, 1254, and 1260 in Male Wistar Rats—Assessment of the Toxic Equivalency Factor (TEF) Approach for Polychlorinated Biphenyls (PCBs)

Comparative Potencies of Aroclors 1232, 1242, 1248, 1254, and 1260 in Male Wistar Rats—Assessment of the Toxic Equivalency Factor (TEF) Approach for Polychlorinated Biphenyls (PCBs). Harris, M., Zacharewski, T., and Safe, S. (1993). Fundam. Appl. Toxicol. 20, 456-463.Immature male Wistar rats were treated with several different doses of the commercial polychlorinated biphenyls (PCBs) Aroclors 1232, 1242, 1248, 1254, and 1260 (10, 40, 160, 480, and 2000 mg/kg) and the effects on body weight gain, thymic atrophy, and the induction of hepatic microsomal aryl hydrocarbon hydroxylase (AHH), ethoxyresorufin O-deethylase (EROD), and pentoxyresorufin O-deethylase (PROD) activities were measured 14 days after treatment. A significant inhibition in body weight gain was observed only in rats treated with high doses of Aroclors 1232 and 1248 and thymic atrophy was not observed for any of the Aroclors. All the Aroclors caused a dose-dependent increase in hepatic microsomal AHH, EROD, and PROD activities. The corresponding ED50 values for the induction of AHH-EROD activities varied from 51 to 678 mg/kg. Aroclor 1260 was the least active inducer of the P4501A1-mediated enzyme activities. In contrast, Aroclor 1260 was a potent inducer of PROD activity (ED50 37 mg/kg), but Aroclors 1232, 1242, 1248, and 1254 did not induce 50% of the maximal response at the highest dose used in this experiment 2000 mg/kg). Previous studies have quantitated the levels of those PCB congeners which induce AHH EROD activities in Aroclors 1232, 1242, 1254, and 1260 and their potencies or toxic equivalency factors (TEFs) relative to that of 2,3,7,8 tetrachlorodibenzo-p-dioxin (TCDD) have also been estimated or experimentally determined. Using highly conservative TEF values it was demonstrated that the calculated ED50s for the Aroclors as inducers of AHH and EROD activity were significantly lower than the observed ED50 values. However, if the TEFs were derived directly from the relative potencies of the PCBs (compared to 2,3,7,8-TCDD) as inducers of AHH and EROD activity, then there was a good correspondence between the observed and calculated ED50 values.

In vivo effect of 7,8-benzoflavone on aryl hydrocarbon hydroxylase activity of mouse liver microsomes

In vivo administration of 3-methylcholanthrene (MC) and 2,3,7,8-tetrachlorodibenzo- p-dioxin (TCDD) produced much higher hepatic microsomal aryl hydrocarbon hydroxylase (AHH) induction than 7,8-benzoflavone (7,8-BF) in both aromatic hydrocarbon (Ah)-responsive and nonresponsive strains of mice. Simultaneous treatment or pre-treatment with 7,8-BF produced an inhibitory effect on AHH induction by MC or TCDD (i.e., the degrees of the inhibition, with TCDD, were 28% in the Ah-responsive strain C57BL/6N (C57) mice and 45% in the nonresponsive strain DDD;Qdj (DDD) mice). However, post-treatment with 7,8-BF was inclined to promote the induction of AHH by MC or TCDD (i.e., the degrees of the enhancement, with MC, were 15% in C57 mice and 45% in DDD mice). These results may suggest that the inhibitory effect of 7,8-BF in vivo is limited not to the combination of AHH inducer (MC or TCDD) but to its application of timing or Ah responsiveness.

BIOMARKERS OF CONTAMINANT EXPOSURE IN COMMON TERNS AND BLACK-CROWNED NIGHT HERONS IN THE GREAT LAKES

Morphological and biochemical indexes of contaminant exposure were examined in hatching common terns (Sterna hirundo) and black-crowned night herons (Nycticorax nycticorax) from industrialized and nonindustrialized locations in the Great Lakes. In 1984, naturally incubated, pipping common tern and black-crowned night heron embryos collected from industrialized locations exhibited smaller femur-length-to-body-weight ratios, elevated hepatic microsomal aryl hydrocarbon hydroxylase (AHH) activities, and lower hepatic DNA concentrations (P < 0.05). In addition, a high incidence of subcutaneous edema was noted in pipping herons (P < 0.01). In 1985, reduced hatching success was observed for laboratory-incubated common tern eggs collected from the industrialized sites, compared to nonindustrialized sites (P < 0.01). Day-old hatchlings exhibited reduced femur-length-to-body-weight ratio, developmental anomalies, and elevated hepatic AHH activity (P < 0.05). For hatching common terns studied in 1984 and 1985, femur-length-to-body-weight ratio was inversely related to AHH activity (r = −0.67, P < 0.05) and inversely related to log-transformed PCB concentrations (r = −0.70, P ≤ 0.05) of unincubated eggs from the same colony. The activity of AHH in hatching terns was also directly related (r = 0.71, P ≤ 0.05) to log-transformed PCB concentrations in unincubated eggs. Other examined contaminants, including DDE, other organochlorine pesticides, and mercury, were not directly related to these effects.

Immunosuppressive and monooxygenase induction activities of polychlorinated diphenyl ether congeners in [formula omitted] mice: Quantitative structure-activity relationships

The dose-response effects of several polychlorinated diphenyl ether (polyCDE) congeners on the inhibition of the splenic plaque-forming cell (PFC) response to sheep red blood cell antigen and the induction of hepatic microsomal aryl hydrocarbon hydroxylase (AHH) and ethoxyresorufin O-deethylase (EROD) activities were determined in male C57BL 6 mice. The immunotoxic potencies for the polyCDE congeners (ED50 values for the suppression of PFCs/spleen and PFCs/10 6 cells) followed the order 2,3,3′,4,4′,5-hexaCDE (0.5 and 2.2 μmol/kg)> 3,3′,4,4′,5-pentaCDE (8.8 and 5.1 μmol/kg) > 2,3′,4,4′,5-pentaCDE (21.8 and 14.2 μmol/kg) > 3,3′,4,4′-tetraCDE (50.6 and 28.7 μmol/kg) > 2,2′,4,4′,5,5′-hexaCDE (81.2 and 56.5 μmol/kg) > 2,2′,4,5,5′-pentaCDE (258 and 228 μmol/kg) > 2,2′,4,4′,5,6′-hexaCDE (>400 μmol/kg for both responses). The potencies of the polyCDE congeners as inducers of hepatic microsomal AHH and EROD activities were similar to their immunotoxicities and only one compound, namely, 2,3′,4,4′,5,5′-hexaCDE, did not cause dose-response immunosuppressive effects in the mice. The structure-activity relationships for the polyCDEs exhibited both differences and similarities. For example, the coplanar 3,3′,4,4′-tetraCDE and 3,3′,4,4′,5-pentaCDE congeners were less immunotoxic than their monoortho 2,3′,4,4′,5-pentaCDE and 2,3,3′,4,4′,5-hexaCDE analogs, respectively, and similar results were also observed for their enzyme induction potencies. For the corresponding polychlorinated biphenyls (PCB) congeners the coplanar compounds were significantly more active than their monoortho analogs. In addition, two diortho-substituted compounds, namely, 2,2′,4,5,5′-pentaCDE and 2,2′,4,4′,5,5′-hexaCDE, were also immunotoxic at a dose of 400 μmol/kg whereas, their PCB analogs were inactive. These studies clearly demonstrate that for the polyCDE congeners, increasing ortho-chloro substitution is less effective in reducing the activity of these congeners compared to the well-recognized ortho effects reported for the PCBs. The differences in the structure-activity relationships between polyCDEs and PCBs are related to the ether bridge in the polyCDEs in which the resultant increased bond length beweeen the two phenyl rings thereby diminishes the effects of ortho substituents on the biochemical and toxic potencies of these compounds.

Structure-dependent induction of aryl hydrocarbon hydroxylase activity in [formula omitted] mice by 2,3,7,8-tetrachlorodibenzo- p-dioxin and related congeners: Mechanistic studies

The time-and dose-dependent induction of murine hepatic microsomal aryl hydrocarbon hydroxylase (AHH) and ethoxyresorufin O-deethylase (EROD) activities by five polychlorinated dibenzo- p-dioxin and dibenzofuran congeners showed that the order of induction potency was 2,3,7,8-tetrachlorodibenzo- p-dioxin (TCDD) > 2,3,7,8-tetrachlorodibenzofuran (PCDF) > 1,2,3,7,8-pentachlorodibenzo- p-dioxin (PCDD) > 1,2,3,7,8-pentachlorodibenzofuran (PCDF) > 2,3,7-trichlorodibenzo- p-dioxin (TrCDD). These structure-induction relationships were comparable to the structure-toxicity and competitive structure-receptor binding relationships previously reported for these compounds. However, using the corresponding radiolabeled congeners, the direct binding K d values for dissociation of the cytosolic receptor-ligand complexes were 9.52, 7.96, 1.27, 3.10, and 8.31 n m for the 2,3,7,8-TCDD, 2,3,7,8-TCDF, 2,3,7-TrCDD, 1,2,3,7,8-PCDD, and 1,2,3,7,8-PCDF congeners and these data were clearly not structure dependent (i.e., similar to the structure-activity relationships). Some of the molecular properties for several radioligand-receptor complexes were similar; for example, the sedimentation coefficients for the cytosolic and nuclear receptor complexes varied from 8.8–10.4 S and 5.98–7.07 S, respectively, and the nuclear receptor complexes for all the radioligands eluted from a DNA-Sepharose column at salt concentrations of 0.27–0.29 m. Treatment of the mice with a maximum inducing dose of 2,3,7,8-[ 3H]TCDD resulted in a time-dependent formation of the nuclear receptor complex which was maximized between 16–24 hr and subsequently decreased up to 72 hr after initial exposure. In parallel studies, the nuclear receptor complex levels were determined 16 hr after treatment of the mice with different doses (2.25, 4.5, and 45 μg/kg) of all five radioligands. The results showed that at submaximal induction of the monooxygenase enzyme activities there was a linear correlation between the induced AHH or EROD activities (after 32 hr) and the corresponding nuclear receptor complex levels. It was also apparent from the data that the relative levels of nuclear receptor complex were structure dependent and this suggests that the transformation or activation of cytosolic receptor complexes may be a ligand structure-dependent process which correlates with the observed structure-activity relationships for 2,3,7,8-TCDD and related compounds.

The structure-dependent effects of heptachlorodibenzofuran isomers in male [formula omitted] mice: Immunotoxicity and monooxygenase enzyme induction

The dose-response effects of the 1,2,3,4,6,7,8-, 1,2,3,4,7,8,9-, 1,2,3,4,6,8,9-, and 1,2,3,4,6,7,9-heptachlorodibenzofurans (HpCDFs) on the splenic plaque-forming cell (PFC) response to sheep erythrocytes and on the induction of hepatic microsomal aryl hydrocarbon hydroxylase (AHH) and ethoxyresorufin- O-deethylase (EROD) activities were determined in male C57 BL 6 mice. The ED50 values for the decrease in the PFCs/spleen, the number of PFCs 10 6 viable cells, and the induction of AHH and EROD activities were 1,2,3,4,6,7,8-HpCDF, 0.011, 0.018, 0.11, and 0.315 μmol/kg, respectively; 1,2,3,4,7,8,9-HpCDF, 0.012, 0.054, 0.70, and 0.20 μmol/kg, respectively; 1,2,3,4,6,7,9-HpCDF, 1.2, 1.3, >43, and >43 μmol/kg, respectively; 1,2,3,4,6,8,9-HpCDF, 1.5, 3.4, 22, and 22 μmol/kg, respectively. It was apparent from these studies that the 2,3,7,8-substituted HpCDF isomers (1,2,3,4,6,7,8- and 1,2,3,4,7,8,9-) were significantly more potent than the compounds which contained only three lateral C1 groups. These results were obtained using a multiple dosing regimen in which 10 separate doses of the HpCDF isomers were administered to the mice by intraperitoneal injection over a period of 12 days. However, when the mice were treated with a single dose of an HpCDF congener, which was equivalent to the total dose used in the multiple dose study, the responses were comparable. A comparison of the relative immunotoxic potencies of the 2,3,7,8-substituted HpCDFs and 2,3,7,8-tetrachlorodibenzo- p-dioxin showed that the latter compound was approximately 10 times more active than the HpCDFs.

Immunosuppressive activities of polychlorinated biphenyls in C57BL/6N mice: Structure-activity relationships as Ah receptor agonists and partial antagonists

The immunosuppressive activity of polychlorinated biphenyl (PCB) congeners is structure-dependent and 2 classes of compounds, namely the coplanar (class I) and monoortho coplanar (class II) congeners exhibit immunotoxicity. This study extends the structure-immunotoxicity relationships for PCBs by investigating representative congeners from the following structural classes of PCBs: monoortho coplanar (2,3,3′,4,4′,5-hexachlorobiphenyl, class II); monoortho coplanar minus a single para-chloro group (2,3,3′4,5,5′-hexachlorobiphenyl, and 2,3,3′,4,5′-pentachlorobiphenyl, class III); diortho coplanar (2,3′,4,4′5′6-hexachlorobiphenyl, class IV); triortho coplanar (2,2′,4,4′,5,6′-hexachlorobiphenyl class V) and a tetraortho-substituted PCB (2,2′,4,4′,6,6′-hexachlorobiphenyl, class VI). The effects of these compounds on the splenic plaque forming cell response to sheep red blood cells was determined in 7–8 week old male C57BL/6N mice. The results showed that the class II–IV congeners were immunotoxic and with only one exception these compounds also induced hepatic microsomal aryl hydrocarbon hydroxylase and ethoxyresorfin O-deethylase activities and displaced [ 3 H]-2,3,7,8- tetrachlorodibenzo-p- dioxin (TCDD) from the cytosolic aryl hydrocarbon (Ah) receptor in competitive binding assays. These results thus extend the structure-activity relationships for PCBs as Ah receptor agonists. The interaction of these PCB congeners with an ED 70–90 dose of TCDD (3.7 nmol/kg) showed that only one structural class of compounds, namely class III, partially antagonized TCDD-mediated immunotoxicity.

The Structure-Dependent Effects of Heptachlorodibenzofuran Isomers in Male C57BL/6 Mice: Immunotoxicity and onooxygenase Enzyme Induction

The Structure-Dependent Effects of Heptachlorodibenzofuran Isomers in Male C57BL/6 Mice: Immunotoxicity and Monooxygenase Enzyme Induction. DICKERSON, R., HOWIE, L., DAVIS, D., AND SAFE, S. (1990). Fundam. Appl. Toxicol. 15,298/307. The dose-response effects of the 1,2,3,4,6,7,8-, 1,2,3,4,7,8,9-, 1,2,3,4,6,8,9-, and 1,2,3,4,6,7,9-heptachlorodibenzofurans (HpCDFs) on the splenic plaque-forming cell (PFC) response to sheep erythrocytes and on the induction of hepatic microsomal aryl hydrocarbon hydroxylase (AHH) and ethoxyresorufin-O-deethylase (EROD) activities were determined in male C57BL/6 mice. The ED50 values for the decrease in the PFCs/spleen, the number of PFCs/10 viable cells, and the induction of AHH and EROD activities were 1,2,3,4,6,7,8-HpCDF, 0.011,0.018,0.11, and 0.315 /imol/kg, respectively; 1,2,3,4,7,8,9-HpCDF, 0.012, 0.054, 0.70, and 0.20 Mmol/kg, respectively; 1,2,3,4,6,7,9-HpCDF, 1.2, 1.3, >43, and >43 Mmol/kg, respectively, 1,2,3,4,6,8,9-HpCDF, 1.5, 3.4, 22, and 22 Mmol/kg, respectively. It was apparent from these studies that the 2,3,7,8-substituted HpCDF isomers (1,2,3,4,6,7,8- and 1,2,3,4,7,8,9-) were significantly more potent than the compounds which contained only three lateral Cl groups. These results were obtained using a multiple dosing regimen in which 10 separate doses of the HpCDF isomers were administered to the mice by intraperitoneal injection over a period of 12 days. However, when the mice were treated with a single dose of an HpCDF congener, which was equivalent to the total dose used in the multiple dose study, the responses were comparable. A comparison of the relative immunotoxic potencies of the 2,3,7,8-substituted HpCDFs and 2,3,7,8-tetrachlorodibenzo-/>-dioxin showed that the latter compound was approximately 10 times more active than the HpCDFs

Stimulation of binding of benzo[ a]pyrene metabolites to DNA by diet-induced peroxidative stress

To investigate the influence of unsaturation of dietary fat on the oxidation of benzo[ a]pyrene-7,8-dihydrodiol to DNA binding products, we fed diets containing 10% by weight of either safflower oil or lard to weanling rats. Compared with the group fed lard, the group fed safflower oil had 2.0- to 2.5-fold higher levels of unstimulated and peroxidation-stimulated activation of benzo[ a]pyrene-7,8-dihydrodiol to DNA-binding metabolites, respectively, in hepatic nuclei. The rats fed safflower oil had a significant 75% higher level of lipid peroxidation as measured by the thiobarbituric acid assay. Rats fed safflower oil also showed 30% greater binding of (−)-benzo[ a]pyrene-7,8-dihydrodiol oxidation products to DNA compared with animals fed lard, following administration of this dihydrodiol enantiomer through the hepatic portal vein. Significant diet-dependent differences were not apparent in DNA binding of the (+)-isomer, or in the tetrol production from either isomer; however, rats fed safflower oil showed a trend towards production of higher levels of anti-benzo[ a]pyrene diol epoxide-derived tetrols. Activities of hepatic nuclear and microsomal aryl hydrocarbon hydroxylase and of cytosolic and microsomal glutathione S-transferases were not significantly affected by diet, nor was the activity of microsome-mediated binding of (+)- or (−)-benzo[ a]pyrene-7,8-dihydrodiol to DNA in vitro. The results indicate that polyunsaturated fat in quantities as low as 10% by weight of the diet is sufficient to increase significantly the extent to which DNA-binding metabolites of benzo[ a]pyrene are produced, and that this increased metabolism is likely to be independent of mixed-function oxidases.

6-substituted-1,3,8-trichlorodibenzofurans as 2,3,7,8-tetrachlorodibenzo- p-dioxin antagonists in the rat: structure activity relationships

The activities of several 6-substituted-1,3,8-trichlorodibenzofurans (CDFs) as partial antagonists of the induction of hepatic microsomal aryl hydrocarbon hydroxylase (AHH) and ethoxyresorufin O-deethylase (EROD) activities in the rat by 2,3,7,8-tetrachlorodibenzo- p-dioxin (TCDD) were structure-dependent. Treatment of the rats with TCDD (16 nmol/kg), the 6-substituted-1,3,8-triCDFs (50 μmol/kg) and TCDD plus the 6-substituted-1,3,8-triCDFs showed that most of the substituted congeners were either inactive (6-methyl, ethyl, propyl, i-propyl, t-butyl) or weak (6-cyclohexyl, nitro) inducers of AHH and EROD activities, whereas TCDD caused and 8.1- and 58-fold induction of these enzyme activities respectively. In the co-administration studies, the 6-methyl, propyl, ethyl, isopropyl and t-butyl analogs partially antagonized the induction of the monooxygenase enzyme activities by TCDD, whereas, the 6-cyclohexyl and 6-nitro-1,3,8-triCDFs exhibited minimal activity as TCDD antagonists. The Ah receptor binding affinities of the 6-substituted compounds were determined in a series of in vitro competitive binding studies using [ 3H]TCDD as the radioligand. Analysis of the data by Scatchard and Dixon plots showed that the avidities for the Ah receptor by the 6-substituted-1,3,8-triCDFs followed the order 6-methyl > 6- t-butyl > 6- i-propyl > 6-propyl ≈ 6-ethyl > 6-cyclohexyl > 6-nitro-1,3,8-triCDF. In addition there was a good correlation between the in vitro binding avidities and K i values for these compounds and their in vivo activity as partial antagonists of the induction of AHH and EROD activities by TCDD. The results suggested that the 6-substituted-1,3,8-triCDFs competitively displaced TCDD from the Ah receptor and this interaction may play a role in the mechanism of action of this class of TCDD antagonists.

Development and validation of in vitro induction assays for toxic halogenated aromatic mixtures: a review.

Halogenated aromatic industrial compounds, typified by the polychlorinated dibenzo-p-dioxins (PCDDs), dibenzofurans (PCDFs) and biphenyls (PCBs) have been identified as residues in almost every component of the global ecosystem. Risk assessment of the complex mixtures of halogenated aromatics found in environmental samples is complicated by analytical problems and the lack of toxicological information on individual compounds and mixtures. Research in our laboratory has focused on the development and vadidation of the in vitro aryl hydrocarbon hydroxylase (AHH) induction assay in rat hepatoma H-4-II E cells in culture for quantitating individual toxic halogenated aryl hydrocarbons and their mixtures. For several PCB, PCDD, PCDF congeners, their mixed bromo/chloro analogs and reconstituted mixtures there was an excellent linear correlation between their -log ED50 values for AHH induction in rat hepatoma cells and their -log ED50 values for in vivo hepatic microsomal AHH induction, inhibition of body weight gain and thymic atrophy in the rat. It has also been shown for selected compounds that there was a good correlation between their in vitro AHH induction potencies and their effects in guinea pigs (AHH induction, inhibition of body weight gain) and mice (immunotoxicity). This assay system has been utilized to quantitative the "2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) equivalents" present in extracts from diverse sources including fly ash from a municipal incinerator and pyrolyzed brominated flame retardants which contain a complex mixture of halogenated dibenzo-p-dioxins and dibenzofurans.

2,3,7,8-Tetrachlorodibenzo- p-dioxin-induced porphyria in genetically inbred mice: Partial antagonism and mechanistic studies

2,3,7,8-Tetrachlorodibenzo- p-dioxin (TCDD) (233 nmol/kg) causes a significant increase of hepatic uroporphyrin, heptacarboxyporphyrin, and total porphyrins in female C57BL/6 mice, ovariectomized C57BL/6 mice, male C57BL/10 mice, and male C57BL/6 mice 3 weeks after treatment. In contrast, 6-methyl-1,3,8-trichlorodibenzofuran (MCDF) was inactive at a dose of 750 μmol/kg. Cotreatment of the mice with TCDD (233 mol/kg) plus MCDF (750 μmol/kg) resulted in partial antagonism of TCDD-induced hepatic porphyrin accumulation only in the female mice. Parallel studies in female C57BL/6 mice showed that the TCDD-induced porphyria was accompanied by the induction of hepatic microsomal aryl hydrocarbon hydroxylase (AHH) and ethoxyresorufin O-deethylase (EROD) activities and the depression of uroporphyrinogen decarboxylase (UROD). MCDF (750 μmol/kg) did not significantly affect these enzymes. In the cotreatment studies (MCDF plus TCDD), MCDF partially antagonized TCDD-induced hepatic porphyrin accumulation but did not affect the levels of hepatic AHH, EROD, or UROD. These results indicate that other factors, in addition to the induction of cytochrome P450-dependent monooxygenases and depressed UROD activity, are important in TCDD-induced porphyria in C57BL/6 female mice.

Hexachlorobenzene: Biochemical effects and synergistic toxic interactions with 2,3,7,8‐tetrachlorodibenzo‐p‐dioxin

Abstract 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 ...

6-methyl-1,3,8-trichlorodibenzofuran (MCDF) as a 2,3,7,8-tetrachlorodibenzo- p-dioxin antagonist in C57BL/6 mice

6-Methyl-1,3,8-trichlorodibenzofuran (MCDF), 2,3,7,8-tetrachlorodibenzo- p-dioxin (TCDD) and TCDD plus MCDF were administered to C57BL/6 mice and their effects on several aryl hydrocarbon (Ah) receptor-mediated responses including hepatic microsomal aryl hydrocarbon hydroxylase (AHH) and ethoxyresorufin O-deethylase (EROD) induction, immunotoxicity and teratogenicity were determined. MCDF did not induce hepatic microsomal AHH and EROD at doses up to 500 μmol/kg, however, co-administration of MCDF (50 μmol/kg) with a dose of TCDD which elicited a submaximal induction response (i.e. ED 80–100, 15 nmol/kg) resulted in some small but significant inhibition of the induction of hepatic microsomal AHH and EROD (14 and 17%, respectively) compared to that observed with TCDD alone. Co-administration of TCDD and other doses of MCDF (10, 100, 200 or 500 μmol/kg) did not effect the induction response. These results were in contrast to the effectiveness of MCDF as an antagonist of the induction of AHH and EROD by TCDD in the rat (up to 50% inhibition of monooxygenase induction). Administration of MCDF (4, 20 and 40 μmol/kg) to C57BL/6 mice caused some inhibition of the splenic plaque-forming cell response to sheep erythrocytes only at the highest dose (26% decrease); the interaction of MCDF (4, 20 and 40 μmol/ kg) and an immunotoxic dose of TCDD (3.7 nmol/kg) resulted in significant protection from the immunotoxic effects of TCDD at the 2 higher dose levels of MCDF. Similarly, MCDF (400 μmol/kg) did not cause cleft palate in mice but at this dose level MCDF afforded some protection from TCDD (20 μg/kg)-mediated cleft palate in mice. However, studies utilizing [ 3H]TCDD suggested that the protective effects may be due to modulation of TCDD reaching the palate in the co-treated animals (MCDF plus TCDD). Although both MCDF and Aroclor 1254 were both weak Ah receptor agonists in C57BL/6 mice, the former compound was much less effective as a TCDD antagonist. The observed species-specific effects for these 2 TCDD antagonists may be related species-dependent differences in receptor structure and receptor-ligand (i.e. agonist or antagonist) interactions/

6-Methyl-1,3,8-trichlorodibenzofuran (MCDF) as a 2,3,7,8-TCDD antagonist in C57BL/6 mice

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 (ED 70–100) and a subeffective dose of MCDF demonstrated that MCDF significantly antagonized 2,3,7,8-TCDD-induced teratogenicity and immunotoxicity in the mice.

Validation of the AHH indoction bioassay for the determination of 2,3,7,8-TCDD toxic equivalents

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 EC 50 values for AHH induction (in vitro) and the in vivo -log ED 50 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 EC 50 (in vitro AHH induction) vs. the -log ED 50 (in vivo responses) and the -log ED 50 (guinea pig in vivo data) vs. -log ED 50 (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.

Characterization of 6-methyl-1,3,8-trichlorodibenzofuran (MCDF) as a 2,3,7,8-TCDD antagonist in male rats: induction of monooxygenases

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 [ 3H]-2,3,7,8-TCDD (+ MCDF), the time-course accumulation of nuclear [ 3H]-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.

Comparative metabolism of (—)benzo[a]pyrene-7,8-dihydrodiol by hepatocytes isolated from two species of bottom-dwelling fish

To investigate the influence of unsaturation of dietary fat on the oxidation of benzo[a]pyrene-7,8-dihydrodiol to DNA binding products, we fed diets containing 10% by weight of either safflower oil or lard to weanling rats. Compared with the group fed lard, the group fed safflower oil had 2.0- to 2.5-fold higher levels of unstimulated and peroxidation-stimulated activation of benzo[a]pyrene-7,8-dihydrodiol to DNA-binding metabolites, respectively, in hepatic nuclei. The rats fed safflower oil had a significant 75% higher level of lipid peroxidation as measured by the thiobarbituric acid assay. Rats fed safflower oil also showed 30% greater binding of (−)-benzo[a]pyrene-7,8-dihydrodiol oxidation products to DNA compared with animals fed lard, following administration of this dihydrodiol enantiomer through the hepatic portal vein. Significant diet-dependent differences were not apparent in DNA binding of the (+)-isomer, or in the tetrol production from either isomer; however, rats fed safflower oil showed a trend towards production of higher levels of anti-benzo[a]pyrene diol epoxide-derived tetrols. Activities of hepatic nuclear and microsomal aryl hydrocarbon hydroxylase and of cytosolic and microsomal glutathione S-transferases were not significantly affected by diet, nor was the activity of microsome-mediated binding of (+)- or (−)-benzo[a]pyrene-7,8-dihydrodiol to DNA in vitro. The results indicate that polyunsaturated fat in quantities as low as 10% by weight of the diet is sufficient to increase significantly the extent to which DNA-binding metabolites of benzo[a]pyrene are produced, and that this increased metabolism is likely to be independent of mixed-function oxidases.

Effect of chronic cysteamine treatment on mouse liver aryl hydrocarbon hydroxylase activity.

Patients receive chronic cysteamine in the management of nephropathic cystinosis. In a previous report our results indicated that acute cysteamine treatment inhibited cytochrome P-450. Cysteamine (85 mg/kg i.p.) was administered daily to female Swiss mice for 1.5 and 8.5 months. Cysteamine treatment (8.5 months) did not affect hepatic microsomal aryl hydrocarbon hydroxylase (AHH) activity compared with controls. A small decrease in liver AHH activity was seen after 1.5 months of treatment with cysteamine. Liver histology, body weight, liver and spleen weights, and serum aminotransferase activity after chronic and subchronic treatment did not differ from controls. Chronic in vivo cysteamine treatment, unlike acute in vitro treatment did not decrease AHH activity. Incubation of isolated murine hepatocytes with cysteamine significantly inhibited AHH activity compared with controls. The inhibition occurred in a concentration-related manner, with 65% inhibition at 8.8 mM (1 mg/mL) (equivalent to the predicted plasma concentration using the maximally tolerable human dose), and 100% inhibition at 44 mM (5 mg/mL). The concentrations used in vitro were not cytotoxic. This suggests that chronic cysteamine treatment may not result in drug interactions and that in vitro results are not always good indicators of in vivo effects.