Aryl Hydrocarbon Hydroxylase Induction Research Articles

Induction of hepatic aryl hydrocarbon hydroxylase activity in rainbow trout ( Salmo gairdneri) exposed to coal liquids

1. 1. The hepatotoxic response of rainbow trout ( Salmo gairdneri) to a middle distillate (MD) and heavy distillate (HD) coal liquid was determined following administration by injection or in food. Hepatic aryl hydrocarbon hydroxylase (AHH) activity was compared to the known AHH inducer, benzo(a)pyrene. 2. 2. Acute toxicities ( ld 50) of 1500 and 1410 mg/kg were obtained 120 hr after intraperitoneal injection (i.p.) for MD and HD coal liquids, respectively. Only the HD induced AHH activity when injected at 5% and 10% of the acute ld 50. 3. 3. Ingestion of 7 or 70 mg HD/kg body weight for 1 week resulted in AHH induction at 5–7 times that of control values. Levels of hepatic AHH activity in fish treated at 7 mg HD/kg declined to 2.6 times control values 7 days after exposure. Maximum induction was observed for the 70 mg HD/kg group that was provided control food for 1 week following 2 weeks of exposure. 4. 4. The hepatic index (HI) or liver to body weight ratio generally increased along with increased activity in the enzyme response system.

DNA transfection of a gene repressing aryl hydrocarbon hydroxylase induction.

High mol. wt genomic DNA from a genetically dominant aryl hydrocarbon hydroxylase (AHH)-deficient mutant cell line derived from the mouse hepatoma cell line Hepa-1 was used to transfect the parent cell line. AHH-deficient transfectants were recovered following single-step selection in medium containing the carcinogen benzo[a]pyrene. The transfectants arose at a frequency of 2 x 10(-7). This frequency was at least 4-fold greater than the frequency of spontaneous forward mutation in this cell line. In another set of experiments, dominant mutant DNA was co-transfected along with the selectable plasmid pSV2ecogpt into parental Hepa-1 cells. The frequency of co-transfection was determined to be 3 x 10(-8). This frequency was approximately 150 times greater than that expected on the basis of coincident but unrelated spontaneous mutation and plasmid uptake. Both types of transfectants were judged, following somatic cell hybridizations, to possess the dominant phenotype of the mutant cell line, demonstrating that a trans-acting dominant negative regulator of AHH was transferred in these experiments. DNA transfection should therefore provide a means for the molecular cloning of the gene that encodes the dominant regulator.

TCDD receptor ligands present in extracts of urban air particulate matter induce aryl hydrocarbon hydroxylase activity and cytochrome P-450c gene expression in rat hepatoma cells

In previous studies we have shown that substances associated with particulates collected from urban air and automobile exhaust bind with high affinity to the 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) receptor present in rat liver cytosol. In this study we used a rat hepatoma cell line, H4IIE, to investigate the effect of such substances on an enzyme system, aryl hydrocarbon hydroxylase (AHH), which is regulated via the TCDD receptor. The results demonstrate that AHH activity in the H4IIE cell line can be induced by extracts of particulates collected from urban air and automobile exhausts in a dose-dependent manner, and that the AHH activity is inducible by five polycyclic aromatic hydrocarbons (PAHs) including 1-/3-nitrobenzo[a]pyrene and 6-chlorochrysene, all present in extracts of particulates from urban air and automobile exhausts. The induction of AHH activity is correlated to apparent TCDD receptor affinity for investigated PAHs (r = 0.85) and particulate extracts. Biochemically, treatment of the cells with 5,6-benzoflavone significantly increased the level of cytochrome P-450c but not P-450d as shown by immunoblotting and analysis of mRNA levels. The data indicate that substances present in extracts of urban air particulates can interact with the TCDD receptor in intact cells and cause an accumulation of cytochrome P-450c mRNA leading to an increased synthesis of the gene product and thus an increase in enzyme activity.

Genotoxicity of various nitroarenes in DNA repair tests with human hepatocytes

The effect of a single topical application of several nitroarenes (1-nitropyrene, nitropyrenes mixture, nitrobenzo(ghi)perylene mixture, 3-nitrofluoranthene, nitrofluoranthene mixture, and nitroperylene mixture) and their corresponding parent arenes to neonatal rats on hepatic and cutaneous drug and carcinogen metabolism was studied. Topical application of each nitroarene (10 mg/kg) resulted in significant induction of aryl hydrocarbon hydroxylase (AHH), 7-ethoxyresorufin O-deethylase (ERD), and 7-ethoxycoumarin O-deethylase (ECD) activities in both skin (1.5- to 14.6-fold) and liver (1.3- to 41.9-fold). The induction of these enzymes by each of the nitroarenes was significant when compared to control or to their corresponding parent arenes. Among the nitroarenes studied, 1-nitropyrene was the least effective in inducing enzyme activities. The inducibility in both skin and liver by different nitroarenes tested was in the following order: nitrofluoranthenes mixture > 3-nitrofluoranthene > nitroperylenes mixture > nitrobenzo(ghi)perylenes mixture > nitropyrenes mixture > 1-nitropyrene. The nitrofluoranthenes mixture and the nitroperylenes mixture were almost as effective as 3-methylcholanthrene (3-MC). Parent arenes were either ineffective or significantly less effective than nitrated arenes in inducing hepatic and/or cutaneous monooxygenase activities. Hepatic and/or cutaneous benzphetamine N-demethylase (BPD), NADPH cytochrome c reductase, NADH ferricyanide reductase activities, and the levels of cytochrome P-450 and cytochrome b5, remained unchanged following treatment with either topically applied nitroarenes or arenes. However, a shift of approximately 1 nm to the blue region in the absorption maximum of hepatic cytochrome P-450 was observed in animals treated with nitroarenes. This shift was not evident in the case of 1-nitropyrene. Analysis of benzo(a)pyrene metabolites by high-pressure liquid chromatography revealed a significant enhancement in the production of metabolites by nitroarene-treated rat skin and liver microsomes. Our studies suggest that nitroarenes are inducers of hepatic and cutaneous monooxygenases in neonatal rats after topical administration and that they resemble the 3-MC type of inducers in this regard.

Electrostatic potentials of some dibenzo-p-dioxins in relation to their biological activities

A computational analysis of the electrostatic potentials of eight halogenated dibenzo-p-dioxins has been carried out at theab initio SCF STO-5G level. It focuses upon the relationships between these potentials and the biological activities of the molecules, including toxicity, aryl hydrocarbon hydroxylase induction and receptor binding. In general, regions of negative potential are found to be associated with the oxygens and with the halogen substituents. Biological activity appears to be related to the presence of an optimum range of negative potentials above the lateral portions of the molecules in conjunction with a weakening of those near the oxygens.

Enzyme induction in rat lung and liver by condensates and fractions from main-stream and side-stream cigarette smoke

Aryl hydrocarbon hydroxylase (AHH) and dimethylnitrosamine demethylase (DMND) activities in pulmonary and hepatic tissues of male Sprague-Dawley rats were assayed following pretreatment with known inducers (benzo[a]pyrene, 3-methylcholanthrene, Aroclor 1254, phenobarbital) and with main-stream (MS) and side-stream (SS) cigarette smoke condensates and their related fractions. Biochemical assays by spectrophotofluorimetry (AHH activity) and spectrophotometry (DMND activity) and by a biological assay (Ames test) were performed to detect AHH and DMND induction. Ames test proved to be much less sensitive than the spectrophotofluorimetric analysis for AHH determination. Both main-stream and side-stream cigarette smoke condensates and some fractions, containing water-soluble bases, water-insoluble bases, and polycyclic aromatic hydrocarbons, were found to induce AHH activity in lung and liver, the lung being induced to the greatest extent. The highest levels of AHH inducibility were found for the SS-smoke condensate and related fractions. In particular, the insoluble bases fractions gave the highest induction. On the contrary, pulmonary DMND activity was not affected by pretreatment with the same materials, while hepatic DMND response was only minimally induced by Aroclor and phenobarbital treatment.

Epidermal cell growth-dependent arylhydrocarbon-hydroxylase (AHH) activity in vitro.

Cytochrome P-450-dependent arylhydrocarbon-hydroxylase (AHH) activity and inducibility by benzanthracene (BA) was measured in cultured guinea pig and human epidermal cells. Basal AHH-activity (AHHb) in guinea pig epidermal cells was much higher than in human epidermal cells. AHHb in guinea pig epidermal cells was directly related to the labeling index and decreased to the original level between the 5th and 7th day of cell culturing. On the other hand, the induction-ratio of AHH reached its maximum level when the number of cells began to rise (proliferation phase) and remained high at day 7 of the cell culture. These results suggest a cell growth dependent activity and inducibility of carcinogen-metabolizing enzymes, such as AHH, in isolated epidermal cells.

Toxic Congener-Specific Analysis of PCBs: Assessment of Toxicity in Equivalents of TCDD

Abstract High resolution capillary gas chromatographic analysis of the polychlorobiphenyls (PCBs) present in snapping turtle eggs, provided quantitative data on selected toxic congeners. The concentrations of these congeners have been converted into equivalent toxic concentrations of 2,3,7,8-tetrachloro-p-dibenzodioxin (TCDD). The toxic equivalent factors (TEFs), necessary to effect this transformation were derived from EC50 values (half the concentration of the toxic congener required to produce the maximum effect) for aryl hydrocarbon hydroxylase (AHH) induction associated with the corresponding toxic PCB congener or isomer. Summation of the resulting toxic equivalents provided a composite assessment of the toxlcity of the PCB mixture in terms of an equivalent concentration of TCDD.

Aroclor 1254 as a 2,3,7,8-tetrachlorodibenzo- p-dioxin antagonist: Effects on enzyme induction and immunotoxicity

2,3,7,8-Tetrachlorodibenzo- p-dioxin (TCDD) and Aroclor 1254 induced the cytochrome P-450 dependent monooxygenases, aryl hydrocarbon hydroxylase (AHH) and ethoxyresorufin O-deethylase (EROD) in rat hepatoma H-4-II E cells and C57BL/6J mice. It has been proposed that both Aroclor 1254 and 2,3,7,8-TCDD induce these enzymes via a common mechanism which features initial binding to the aryl hydrocarbon (Ah) cytosolic receptor protein. The major difference between these compounds was the relative potency (i.e. 2,3,7,8-TCDD ⪢ Aroclor 1254). Cotreatment of rat hepatoma H-4-II E cells or C57BL/6J mice with a dose of 2,3,7,8-TCDD which submaximally induces AHH and EROD and a dose of Aroclor 1254 which exhibited little or no induction activity resulted in significant antagonism of the induction effect of 2,3,7,8-TCDD. For example, cotreatment of C57BL/6J mice with 2,3,7,8-TCDD (15 nmol/kg) and Aroclor 1254 (25, 75 and 150 μmol/kg) resulted in up to 23% antagonism of AHH induction by 2,3,7,8-TCDD. Moreover, cotreatment with a higher dose of the 2,3,7,8-TCDD agonist (30 or 50 nmol/kg) partially reversed some of the antagonism by Aroclor 1254. In vivo antagonism was observed only at Aroclor 1254/2,3,7,8-TCDD molar ratios of 1667:1, 5000:1 and 10 000:1. Administration of 2,3,7,8-TCDD (3.72 nmol/kg) to C57BL/6J mice resulted in a 76% decrease in the splenic plaque forming cell response to sheep red blood cells. This T-cell mediated immunotoxic effect of 2,3,7,8-TCDD segregates with the Ah locus. In contrast, administration of 5, 15, 75 and 150 μmol/kg of Aroclor 1254 resulted in impairment of the immune response only at the highest dose level. However, cotreatment of mice with 2,3,7,8-TCDD (3.72 nmol/kg) and Aroclor 1254 (5, 15 or 75 μmol/kg) resulted in no significant decrease in the plaque forming cell response and complete protection from the immunotoxicity of 2,3,7,8-TCDD. Cotreatment of the mice with Aroclor 1254 (75 μmol/kg) and a higher dose of the 2,3,7,8-TCDD agonist resulted in partial reversal of the protective effects of Aroclor 1254. The in vitro and in vivo data suggest that within specific antagonist/agonist dose ratios, Aroclor 1254 can antagonize at least 2 Ah receptor-mediated effects of 2,3,7,8-TCDD, namely AHH induction and immunotoxicity.

2,3,7,8‐Tetrachlorodibenzo‐p‐dioxin receptors regulate transcription of the cytochrome P1‐450 gene

The environmental contaminant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) dioxin, produces a diverse set of biological responses which, in some cases, reflects the altered expression of specific genes. An intracellular receptor protein binds TCDD saturably and with high affinity and mediates several of TCDD's biological effects. In mouse hepatoma cells, TCDD induces aryl hydrocarbon hydroxylase activity by activating the transcription of the cytochrome P1-450 gene. Studies of receptor-defective variant cells indicate that the activation of cytochrome P1-450 gene transcription requires functional TCDD receptors. Analysis of the DNA that flanks the 5'-end of the mouse cytochrome P1-450 gene reveals at least three control regions: a promoter, an inhibitory element, and a dioxin-responsive element (DRE). Therefore, expression of the cytochrome P1-450 gene represents a balance between negative and positive control. The DRE contains two discrete, non-overlapping DNA domains that respond to TCDD. Each TCDD-responsive domain acts independently of the other, each requires TCDD receptors for function, and each has the properties of a transcriptional enhancer. For example, the function of the DREs is relatively independent of both their location and their orientation with respect to the promoter. Together, the DREs and the TCDD-receptor complex constitute a dioxin-responsive enhancer system. Exposure of cells to TCDD results in the protection of a specific DNA domain from exonuclease digestion. This protection requires TCDD receptors. The protected domain maps to a DRE. This observation implies that the TCDD-receptor complex interacts with the DRE to activate the transcription of the cytochrome P1-450 gene.

Pulmonary aryl hydrocarbon hydroxylase activity of mice, rats and guinea pigs following long term exposure to mainstream and sidestream cigarette smoke

Male, C57BL mice, Sprague-Dawley rats and Hartley guinea pigs were given daily exposure to either mainstream or sidestream whole smoke from University of Kentucky Reference cigarette, 2R1, for 16 weeks. Pulmonary aryl hydrocarbon hydroxylase (AHH) activity was assayed in lung tissue 9000 g supernatants by measuring the conversion of [ 3H] benzo [ a] pyrene to its various metabolites. Both mainstream as well as sidestream smoke exposure increased the pulmonary AHH activity by approximately 3–4-fold over room and sham control rats and mice, but not guinea pigs. The magnitude of AHH induction in sidestream- and mainstream-exposed animals was similar, even though the dose of smoke particulates received by rats and mice in sidestream-exposed groups was significantly less than the amount inhaled by their corresponding mainstream-exposed groups. The data suggest that like mainstream, the sidestream cigarette smoke is also capable of inducing pulmonary AHH in responsive animal species.

Polybrominated dibenzo- p-dioxins and related compounds: Quantitative in vivo and in vitro structure-activity relationships

The effects of structure on the in vitro receptor binding affinities, aryl hydrocarbon hydroxylase (AHH) and ethoxyresorufin O-deethylase (EROD) induction potencies in rat hepatoma cells were determined for the following compounds: 2-bromo-, 2,7/2,8-dibromo-, 2,3,7-tribromo-, 2,4,6,8/1,3,7,9-tetrabromo-, 2,3,7,8-tetrabromo-, 1,3,7,8-tetrabromo-, 1,2,3,7,8-pentabromo-, 1,2,4,7,8-pentabromo-, 2,3,-dibromo-7,8-dichloro-, 2,8-dibromo-3,7-dichloro- and 2-bromo-3,7,8-trichlorodibenzo- p-dioxin. The structure-activity relationships (SARs) for the polybrominated dibenzo- p-dioxins (PBDDs) were comparable for both in vitro responses: the most active compounds were substituted only in the lateral 2,3,7 and 8 position and the addition of non-lateral or removal of lateral halogen substituents reduced the activity of the resultant compound. The biologic and toxic effects of 2,3,7,8-tetrabromo-, 1,3,7,8-tetrabromo-, 1,2,4,7,8-pentabromo-1,2,3,7,8-pentabromo-, 2-bromo-3,7,8-trichloro- and 2,3-dibromo-7,8-dichlorodibenzo- p-dioxin on several receptor-mediated responses (thymic atrophy, body weight loss, hepatic microsomal AHH and EROD induction) were determined in a dose-response fashion in immature male Wistar rats. A comparison of the ED 50 values for the in vivo responses demonstrated that the SARs for the PBDDs and brominated polychlorinated dibenzo- p-dioxins were comparable to those observed for in vitro receptor binding and AHH induction. Moreover, there was an excellent linear correlation between the -log EC 50 (in vitro AHH induction) vs. the in vivo -log ED 50 (thymic atrophy) and -log ED 50 (body wt loss) correlation coefficient, r = 0.97 for all 2 correlations).

Mechanism of cytotoxicity of aflatoxin B 1: Role of cytochrome P 1-450

A mouse hepatoma cell line, Hepa-1, is highly sensitive to the toxic effects of Aflatoxin B 1 (AFB 1). Half maximal survival (LD 50) of cells occurs at 0.068 ug AFB 1/ml. Benzo(a)anthracene, which induces aryl hydrocarbon hydroxylase and cytochrome P 1-450 in Hepa-1, causes a slight increase in the toxicity of AFB 1 (LD 50 = 0.034 ug/ml). An aryl hydrocarbon hydroxylase- and cytochrome P 1-450-deficient mutant of Hepa-1 is, however, over 100 times more resistant to AFB 1 than Hepa-1. Almost no decline in survival is observed at 5 ug AFB 1/ml. Cytochrome P 1-450 thus effects strongly on the cytotoxicity of AFB 1 in these cells. The basal activity in Hepa-1 is enough to elicit an almost full toxic effect. AFB 1, although a substrate for cytochrome P 1-450, does not act as an inducer of aryl hydrocarbon hydroxylase.

Dose-dependent elevation of Ah receptor binding by TCDD in rat liver

Changes in [ 3H]TCDD binding to unoccupied liver Ah receptor were examined following chronic or acute administration of 2,3,7,8-tetrachlorodibenzo- p-dioxin (TCDD) to adult female Sprague-Dawley rats. Chronic biweekly administration of TCDD equivalent to 0, 10, 30, or 100 ng/kg/day TCDD for 22 weeks caused dose-dependent increases in liver TCDD concentration, aryl hydrocarbon hydroxylase (AHH) induction, and [ 3H]TCDD binding to unoccupied cytosolic Ah receptor sites. Maximal increases (twofold) of cytosolic receptor binding occurred at an estimated dose of slightly greater than 30 ng/kg/day. The increase in [ 3H]TCDD binding was half-maximal at an estimated dose of approximately 17 ng/kg/day which produced a liver concentration of 1.5 ppb TCDD. Cytosolic [ 3H]TCDD binding in control and treated animals sedimented mainly in the 8–9 S region of sucrose density gradients with a minor peak sedimenting in the 4–5 S region. Binding was markedly elevated in the 8–9 S region of cytosols from the TCDD-induced rats; however, TCDD treatment had no affect on [ 3H]TCDD binding in the 4–5 S region. Saturation and Scatchard analyses of Ah receptor binding showed no apparent changes in K d following chronic TCDD treatment; however, a twofold increase in the number of unoccupied Ah receptor binding sites was observed. Neither aging nor ovariectomy significantly changed measurable cytosolic Ah receptor binding in control animals. When adult female rats were administered a single dose of TCDD (6 μg/kg) an initial drop (∼40%) in cytosolic receptor binding was observed at 30 and 60 min, followed by a steady increase in binding up to 250% of controls 9 days after TCDD treatment.

Vitamins E and K induce aryl hydrocarbon hydroxylase activity in human cell cultures

Two fat soluble vitamins, Vitamins E and K, when added into culture medium, were found to increase aryl hydrocarbon hydroxylase activity in human cultured cells. The extent of induction in a hepatoma-derived cell line (Hep G2) by these vitamins is of similar magnitude to those cells receiving benz[a]anthracene; whereas in a mammary tumor-derived cell line (MCF-7), benz[a]anthracene is the best inducer for the hydroxylase activity. The increase of the hydroxylase activity is associated with increased levels of a specific mRNA coding for polynuclear aromatic hydrocarbons-induced form of cytochrome P-450 with Vitamins E and K treatment. The size of the induced mRNA is 3.3 kilobase which is the same as that of benz[a]anthracene treatment.

Binding of polynuclear aromatic hydrocarbons to the rat 4S cytosolic binding protein: Structure-activity relationships

The relative competitive binding affinites of benzo[ a]pyrene (B[ a]P), benzo[ e]pyrene, benzo[ g,h,i]perylene, picene, 7,12-dimethylbenz[ a]anthracene, 1,2,3,4-dibenz[ a]anthracene, 1,2,5,6-dibenz[ a]anthracene, perylene, 4 H-cyclopenta[ d,e,f]-phenanthrene, benz[ a]anthracene, triphenylethylene and triptycene for the rat hepatic cytosolic 4S binding protein were determined using [ 3H] benzo[ a]pyrene as the radioligand. With the exception of triphenlethylene, triptycene and 4 H-cycolopenta[ d,e,f]phenanthrene, the EC 50 values for the remainder of these compounds were between 1.25 × 10 −7 and 2.5 × 10 −8 M with 1,2,5,6-dibenz[ a]anthracene being the most active ligand. A comparison of the relative cytosolic Ah (9S) receptor binding affinities and aryl hydrocarbon hydroxylase (AHH) induction potencies of these hydrocarbons with their 4S protein binding affinities demonstrated the following: (a) five compounds, namely 1,2,5,6-dibenz[ a]anthracene, 1,2,3,4-dibenz[ a]anthracene, picene, benzo[ a]pyrene and 3-methylcholanthrene exhibited high to moderate binding affinities for the 4S and 9S cytosolic proteins (EC 50 values < 10 −6 M) and induced AHH in rat hepatoma cells; (b) three compounds, namely perylene, benzo[ e]pyrene and benzo[ g,h,i]perylene exhibited high affinities for the 4S binding protein (1.25 × 10 −7, 4.4 × 10 −8 and 2.9 × 10 −8 M, respectively) and low affinities (EC 50 values 10 −5 M) for the Ah receptor protein; (c) moreover these three compounds did not induce AHH in rat hepatoma H-4-II E cells in culture. These data suggest that the 4S binding protein may not play a significant role in AHH induction although the results do not rule out a function for this protein in the transregulation of AHH and its associated cytochromes P-450.

Transcriptional control of human cytochrome P1-450 gene expression by 2,3,7,8-tetrachlorodibenzo- p-dioxin in human tissue culture cell lines

2,3,7,8-Tetrachlorodibenzo- p-dioxin (TCDD) induces aryl hydrocarbon hydroxylase (AHH) activity and human cytochrome P 1-450 mRNA in the human breast carcinoma MCF-7 and hepatoblastoma HepG 2 tissue culture cell lines. Although AHH activities induced by 100 n m TCDD are comparable in these cell lines, the EC 50 values for TCDD differ: EC 50 ≅ 1 n m for HepG 2; EC 50 > 20 n m for MCF-7. In order to determine the mechanism responsible for this difference in EC 50, we have examined putative regulatory factors such as the intracellular TCDD receptor as well as the kinetics of mRNA transcription and accumulation in these cells. TCDD increases transcription of hP 1450 mRNA in both MCF-7 and HepG 2 cells; however, MCF-7 cells require higher concentrations of TCDD to produce transcriptional activation comparable to that observed for HepG 2 cells. These data indicate that the difference in EC 50 is determined at an early step in the induction of hP 1450 mRNA. With the use of a sensitive assay based on high-performance anion-exchange liquid chromatography, an intracellular protein which binds TCDD with high affinity was detected in HepG 2 cytosolic fractions but not in MCF-7 cells. Thus, the difference in EC 50 for TCDD can be correlated to the differences in TCDD binding. We postulate that MCF-7 cells contain a “defective” receptor with decreased affinity for TCDD.

Embryotoxicity, teratogenicity, and aryl hydrocarbon hydroxylase activity in forster's terns on green bay, lake michigan

Known reproductive problems, including congenital malformations and poor hatching success, exist for the state endangered Forster's tern (Sterna forsteri) in Green Bay, Wisconsin. Twenty Forster's tern eggs were collected from separate nests at a natural colony with documented reproductive problems, situated at Green Bay, Lake Michigan, and an inland colony at Lake Poygan (control) where reproduction was documented as normal. Eggs from the two locations were placed in the same laboratory incubator and candled throughout incubation. Hatching success of Green Bay eggs was 52% of that for controls. Several early embryonic deaths occurred, but most mortality occurred close to the time of hatching. Liver microsomal aryl hydrocarbon hydroxylase activity was elevated approximately threefold in Green Bay hatchlings compared to controls. Green Bay terns that hatched weighed less than controls, had an increased liver to body weight ratio, and had a shorter femur length. Two Green Bay embryos that failed to hatch had anomalies, one with a crossed beak and one with poor ossification of the foot. One Green Bay hatchling had an abnormally ossified ilium. These effects were observed in eggs where there were measureable levels of aryl hydrocarbon hydroxylase inducers including polychlorinated biphenyls and polychlorinated dibenzo-p-dioxins.

Determination of 2,3,7,8-TCDD toxic equivalent factors (TEFs): support for the use of the in vitro AHH induction assay

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 effects of receptor antagonists on the AHH induction activity of 2,3,7,8-TCDD in C57BL/6 and DBA/2 mice: 1,3,6,8-tetrachlorodibenzofuran

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.