Role of AHR2 in the expression of novel cytochrome P450 1 family genes, cell cycle genes, and morphological defects in developing zebra fish exposed to 3,3',4,4',5-pentachlorobiphenyl or 2,3,7,8-tetrachlorodibenzo-p-dioxin.

Halogenated agonists for the aryl hydrocarbon receptor (AHR), such as 3,3',4,4',5-pentachlorobiphenyl (PCB126) and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), cause developmental toxicity in fish. AHR dependence of these effects is known for TCDD but only presumed for PCB126, and the AHR-regulated genes involved are known only in part. We defined the role of AHR in regulation of four cytochrome P450 1 (CYP1) genes and the effect of PCB126 on cell cycle genes (i.e., PCNA and cyclin E) in zebra fish (Danio rerio) embryos. Basal and PCB126-induced expression of CYP1A, CYP1B1, CYP1C1, and CYP1C2 was examined over time as well as in relation to cell cycle gene expression and morphological effects of PCB126 in developing zebra fish. The four CYP1 genes differed in the time for maximal basal and induced expression, i.e., CYP1B1 peaked within 2 days postfertilization (dpf), the CYP1Cs around hatching (3 dpf), and CYP1A after hatching (14-21 dpf). These results indicate developmental periods when the CYP1s may play physiological roles. PCB126 (0.3-100nM) caused concentration-dependent CYP1 gene induction (EC50: 1.4-2.7nM, Lowest observed effect concentration [LOEC]: 0.3-1nM) and pericardial edema (EC50: 4.4nM, LOEC: 3nM) in 3-dpf embryos. Blockage of AHR2 translation significantly inhibited these effects of PCB126 and TCDD. PCNA gene expression was reduced by PCB126 in a concentration-dependent manner, suggesting that PCB126 could suppress cell proliferation. Our results indicate that the four CYP1 genes examined are regulated by AHR2 and that the effect of PCB126 on morphology in zebra fish embryos is AHR2 dependent. Moreover, the developmental patterns of expression and induction suggest that CYP1 enzymes could function in normal development and in developmental toxicity of PCB126 in fish embryos.     

Uncoupling of cytochrome P450 1A and stimulation of reactive oxygen species production by co-planar polychlorinated biphenyl congeners

The non-ortho-polychlorinated biphenyl (PCB) congener 3,3'4,4'-tetrachlorobiphenyl (PCB 77) can uncouple the catalytic cycle of fish (scup) cytochrome P4501A (CYP1A) and mammalian (rat, human) CYP1A1, stimulating release of reactive oxygen species (ROS). PCB 77 also inactivates CYP1A in an NADPH-, oxygen-, and time-dependent process, linked to uncoupling. We addressed a hypothesis that planar halogenated hydrocarbons generally will uncouple CYP1A. Thus, additional PCB congeners including non-ortho-3,3',4,4',5'-pentachlorobiphenyl (PCB 126) and 3,3',4,4',5,5'-hexachlorobiphenyl (PCB 169), mono-ortho-2,3,3',4,4'-pentachlorobiphenyl (PCB 105) and di-ortho-2,2',5,5'-tetrachlorobiphenyl (PCB 52), as well as the polycyclic aromatic hydrocarbon benzo[a]pyrene (B[a]P), were examined for their ability to stimulate microsomal ROS production and to inactivate CYP1A. Incubated without NADPH, non-ortho-PCB 126 and -PCB 169 both inhibited microsomal CYP1A activity (ethoxyresorufin O-deethylase; EROD). When NADPH was included, these congeners caused a progressive inactivation of CYP1A, in addition to the inhibition. The determined K(Inact) values for inactivation were 0.14 and 0.08 microM, respectively, for PCB 126 and PCB 169, similar to the 0.05 microM for PCB 77 previously reported. The mono-ortho-PCB 105 weakly inhibited and weakly inactivated CYP1A. The di-ortho-PCB 52 neither inhibited nor inactivated CYP1A. Alone, B[a]P strongly inhibited CYP1A, but when NADPH was added that inhibition was reversed, apparently by metabolic depletion of the substrate, and there was no inactivation. PCB 126 and PCB 169 stimulated release of ROS from induced liver microsomes, while B[a]P, PCB 52 and PCB 105 did not. ROS release and CYP1A inactivation stimulated by the non-ortho-PCB 126 and PCB 169 indicate an uncoupling of CYP1A like that previously shown with PCB 77. The uncoupling and release of ROS further suggest a participation of CYP1A in the oxidative stress associated with some planar halogenated aryl hydrocarbon receptor agonists.     

Effects of 3,3',4,4',5-pentachlorobiphenyl, a coplanar polychlorinated biphenyl congener, on cultured neonatal mouse testis.

3,3',4,4',5-Pentachlorobiphenyl (PCB126), a congener with a planar configuration, has been established to have relatively strong toxicities similar to those of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) via aryl hydrocarbon receptors. We investigated the effects of this coplanar PCB on mammalian early spermatogenesis and steroidogenesis in a mouse neonatal testicular organ culture system. Testes collected from newborn mice were subjected to organ culture in medium containing 0, 10, 100 or 1000 nM PCB126. Histochemical analysis revealed that the BrdU-labeling indices of both spermatogenic cells and Sertoli cells were unchanged in all testis specimens exposed to the coplanar PCB. CYP1A1 and steroidogenic enzymes (P450scc, P450c17, 3beta-HSD and 17beta-HSD) mRNA levels were determined by semiquantitative RT-PCR. The CYP1A1 mRNA level in cultured testis was significantly increased by PCB126 in a dose-dependent manner. Although mRNA levels of 3beta-HSD and 17beta-HSD were unchanged, the P450scc mRNA level was significantly down-regulated by PCB126 in a dose-dependent manner. In contrast, the P450c17 mRNA level was significantly higher in 1000 nM PCB126-exposed testis than in control testis. These results suggest that the coplanar PCB does not alter the proliferative activity of spermatogenic cells and Sertoli cells in neonatal testis, but that it directly affects the expression of steroidogenic enzyme genes.     

The anti-estrogenicity of Ah receptor agonists in carp (Cyprinus carpio) hepatocytes.

Cultured hepatocytes of female carp (Cyprinus carpio) were coexposed for 4 days to 200 nM 17beta-estradiol (E2), and concentration ranges of nine known Ah receptor (AhR) agonists: 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), 3,3'4,4'5-pentachlorobiphenyl (PCB 126), 2,3'4,4'5-pentachlorobiphenyl (PCB 118), beta-naphthoflavone (BNF), benzo(a)pyrene (BaP), benzo(a)anthracene (BaA), diindolylmethane (DIM), 6-methyl-1,3,8-trichlorodibenzofuran (MCDF) and hexachlorobenzene (HCB). TCDD caused a greater than 100-fold induction of cytochrome P4501A (CYP1A) activity, measured as ethoxyresorufin O-deethylase (EROD), with an EC50 of 6 pM. Based on EC50 values, the order of potency as CYP1A inducers was TCDD > PCB 126 > BNF > BaP > BaA > PCB 118. DIM and MCDF caused a lower maximum CYP1A induction (< 9-fold), whereas HCB caused no EROD induction at concentrations up to 6 microM. TCDD, PCB 126, BNF, BaP, and DIM also caused a concentration-dependent suppression of the secretion of the yolk protein vitellogenin (Vtg), relative to E2-treated hepatocytes. Suppression of Vtg secretion was not directly correlated with EROD activity, and the antiestrogenic effects occurred at higher concentrations than the induction of CYP1A. This indicates that the anti-estrogenicity was not caused by increased metabolism of E2 due to induction of CYP1A. Nevertheless, the order of potency of the tested compounds for suppression of Vtg secretion was comparable to the order of potency for CYP1A induction. This concurrence suggests that the anti-estrogenicity of these compounds is AhR-mediated, but does not involve CYP1A. This could be relevant for feral fish populations, as they are frequently exposed to AhR agonists, to an extent that AhR-mediated effects are observed.     

Dietary accumulation and biochemical responses of juvenile rainbow trout (Oncorhynchus mykiss) to 3,3',4,4',5-pentachlorobiphenyl (PCB 126)

Juvenile rainbow trout (Oncorhynchus mykiss) (initial weights 2-5 g) were exposed to three dietary concentrations (0, 12.4 and 126 ng g(-1), wet weight) of a 14C-labelled 3,3',4,4',5-pentachlorobiphenyl (PCB 126) for 30 days followed by 160 days of clean food. We assessed bioaccumulation, histology (liver and thyroid) and biochemical responses (liver ethoxyresorufin-O-deethylase (EROD), liver vitamins (retinoids and tocopherol) and muscle thyroid hormone levels) along with growth and survival. The half-life of PCB 126 in the rainbow trout ranged from 82 to 180 days while biomagnification factors (BMF) ranged from 2.5 to 4.1 providing further evidence that PCB 126 is among the most bioaccumulative PCB congeners. Toluene extractable 14C declined with time in the trout suggesting the possibility of some biotransformation and/or covalent bonding with biological macromolecules. The threshold for liver EROD induction by PCB 126 was approximately 0.1 ng g(-1) (wet weight). EROD activities in the low- and high treatments were 9 and 44 times greater than control, respectively, and remained elevated throughout the experiment. EROD activity was correlated with whole body concentrations of PCB 126 although there was evidence of EROD activity suppression in the highly exposed fish. Liver didehydroretinoids and tocopherol concentrations were depressed by the high PCB 126 dose after 30 days exposure. Initially, muscle concentrations of thyroxine (T4) and triiodo-L-thyronine (T3) declined as the fish grew during the experiment, and exposure to PCB 126 accelerated the growth related decline. More information is needed to assess the functional significance of the reduced muscular stores of thyroid hormones. Despite the changes in liver EROD, liver vitamins and muscle thyroid hormones, liver and thyroid histology in trout examined after 30 days exposure and growth parameters were unaffected by PCB 126. This indicates that the functional competences of the physiological factors associated with growth were maintained under the experimental conditions.     

CYP1A-dependent activation of xenobiotics in endothelial linings of the chorioallantoic membrane (CAM) in birds

Metabolic activation of the heterocyclic amine 3-amino -1,4-dimethyl-5 H-pyrido[4,3-b]indole (Trp-P-1) and 7-ethoxyresorufin O-deethylase (EROD) activity were examined in the chorioallantoic membrane (CAM) of 15-day-old chicken and 18-day-old eider duck embryos. The embryos were pretreated with an Ah receptor agonist, i.e. beta-naphthoflavone (BNF) or 3,3',4,4',5-pentachlorobiphenyl (PCB 126), or vehicle in ovo. BNF and PCB 126 induced EROD activity and covalent binding of [3H]Trp-P-1 seven- to tenfold in the CAM of chicken embryos. In the CAM of eider duck embryos, which are known to be nonresponsive to coplanar PCBs, PCB 126 treatment had no effect on EROD activity or covalent binding of [3H]Trp-P-1 whereas BNF treatment increased these activities five- and threefold, respectively. Light microscopic autoradiography was used to identify the cellular localization of covalent binding of [3H]Trp-P-1 in the CAM. Preferential binding was observed in endothelial cells in intraepithelial capillaries in the chorionic epithelium and in blood vessels in the mesenchymal layer. The addition of the CYP1A inhibitor ellipticine abolished the covalent binding of [3H]Trp-P-1 in the CAM of BNF- and PCB 126-treated chicken and eider duck embryos. The results suggest that CYP1A-dependent metabolic activity can be induced in blood vessel endothelia in the CAM of bird embryos following exposure to Ah receptor agonists and that the CAM may be a target tissue for CYP1A-activated environmental pollutants. Furthermore, the highly vascularized CAM could be used as a model for studies of Ah receptor-mediated alterations in the vasculature.     

Altered thyroid status in lake trout (Salvelinus namaycush) exposed to co-planar 3,3',4,4',5-pentachlorobiphenyl

Recent studies indicate that co-planar 3,3',4,4',5-pentachlorobiphenyl (PCB) congeners or their metabolites may disrupt thyroid function in fishes. Although co-planar PCB have been detected at microgram per kilogram levels in fish from contaminated areas, few studies have examined mechanisms whereby, co-planar PCBs may alter thyroid function in fish. We treated immature lake trout by intraperitoneal (i.p.)-injection or dietary gavage with vehicle containing 0, 0.7, 1.2, 25 or 40 microg 3,3',4,4',5-pentachlorobiphenyl (PCB 126) per kgBW. Blood and tissue samples were collected at various times up to 61 weeks following exposure. The treatments produced sustained dose-dependent elevations of tissue (PCB 126) concentrations. Thyroid epithelial cell height (TECH), plasma thyroxine (T4) and 3,3',5-triiodo-l-thyronine (T3) concentrations, hepatic 5'-monodeiodinase, hepatic glucuronidation of T4 and T3, as well as plasma T4 kinetics and fish growth were analyzed. Exposure to the highest doses of PCB 126 caused increased TECH, plasma T4 dynamics and T4-glucuronidation (T4-G). PCB 126 did not affect 5'-monodeiodinase and T3-glucuronidation (T3-G) and there were no effects on fish growth or condition. Because T3 status and growth were unaffected, the thyroid system was able to compensate for the alterations caused by the PCB 126 exposure. It is clear that concentrations of co-planar PCBs similar to those found in predatory fish from contaminated areas in the Great Lakes are capable of enhancing metabolism of T4. These changes may be of significance when T4 requirements are high for other reasons (e.g. periods of rapid growth, warm temperatures, metamorphosis, and parr-smolt transformation).     

Resistance to Cyp3a induction by polychlorinated biphenyls,including non-dioxin-like PCB153, in gills of killifish (Fundulus heteroclitus) from New Bedford Harbor

Previous reports suggested that non-dioxin-like (NDL) PCB153 effects on cytochrome P450 3A (Cyp3a) expression in Atlantic killifish (Fundulus heteroclitus) gills differed between F0 generation fish from a PCB site (New Bedford Harbor; NBH) and a reference site (Scorton Creek; SC). Here, we examined effects of PCB153, dioxin-like (DL) PCB126, or a mixture of both, on Cyp3a56 mRNA in killifish generations removed from the wild, without environmental PCB exposures. PCB126 effects in liver and gills differed between populations, as expected. Gill Cyp3a56 was not affected by either congener in NBH F2 generation fish, but was induced by PCB153 in SC F1 fish, with females showing a greater response. PCB153 did not affect Cyp3a56 in liver of either population. Results suggest a heritable resistance to NDL-PCBs in killifish from NBH, in addition to that reported for DL PCBs. Induction of Cyp3a56 in gills may be a biomarker of exposure to NDL PCBs in fish populations that are not resistant to PCBs.     

Inductive and inhibitory effects of non-ortho-substituted polychlorinated biphenyls on estrogen metabolism and human cytochromes P450 1A1 and 1B1.

The effects of a series of non-ortho-substituted polychlorinated biphenyls (PCBs) on human cytochrome P450 1A1 (CYP1A1), a 17beta-estradiol (E2) 2-hydroxylase, and P450 1B1 (CYP1B1), an E2 4-hydroxylase, were investigated in HepG2 and MCF-7 cells. Elevated rates of 2- and 4-methoxyestradiol (2- and 4-MeOE2) formation in PCB-treated cultures were measured as activities of CYP1A1 and CYP1B1, respectively. Of the congeners investigated, 3,4,4',5-tetrachlorobiphenyl (PCB 81), 3,3',4,4',5-pentachlorobiphenyl (PCB 126), and 3,4',5-trichlorobiphenyl (PCB 39) caused marked stimulation of E2 metabolism in both cell lines. Northern blot analyses confirmed that exposure of MCF-7 cells to PCBs 81, 126, and 39 caused highly elevated levels of the CYP1A1 and CYP1B1 mRNAs. Exposure of MCF-7 cells to 3,3',4,4',5,5'-hexachlorobiphenyl (PCB 169) resulted in elevated levels of the CYP1A1 and CYP1B1 mRNAs, but did not cause elevated rates of E2 metabolism; rather, 4-MeOE2 production was depressed to below control levels in PCB 169-treated cultures. PCB 169 also inhibited the 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-induced 4-MeOE2 and, to a lesser extent, 2-MeOE2 production in MCF-7 cells, as did PCB 126 and several other congeners. In microsomal assays, inhibition of cDNA-expressed human CYP1B1 by PCBs 169 and 126 was demonstrated. These studies with one subgroup of PCBs, the non-ortho-substituted congeners, underscore the complexity and diversity of effects of PCBs, as individual congeners were found both to induce expression and to inhibit activity of human CYP1B1 and CYP1A1.     

A non-destructive BFCOD assay for in vivo measurement of cytochrome P450 3A (CYP3A) enzyme activity in fish embryos and larvae

There is increasing interest in quantifying the exposure and effects of anthropogenic contaminants in fish. Determination of exposures in wild fish is routinely performed, but methods to investigate potential effects are less established. One of the most relevant approaches would be the use of in vivo assays, but existing assays are often limited to in vitro determination of enzyme activity. Many pharmaceuticals and some persistent pollutants activate, and are metabolized by cytochrome P4503A (CYP3A), which make it a relevant and desirable target for biomarker research. We altered the established 7-benzyloxy-4-trifluoromethylcoumarin-O-debenzylation (BFCOD) in vitro protocol for CYP3A activity determination, developing a rapid and inexpensive method to measure in vivo (and in ovo) CYP3A activity in two fish systems: Gulf killifish (Fundulus grandis) and zebrafish (Danio rerio) early life stages. Even with very low concentrations of 7-benzyloxy-4-trifluoromethyl coumarin (BFC, 0.06?µM or 20?µg/L), we were able to detect significant induction in CYP3A activity in embryos of F. grandis, as well as in larvae of D. rerio in response to benzo[a]pyrene (BaP) and fluoranthene (FL) exposures. Because of concerns regarding the possible contribution of CYP1A to BFCOD activity from previous research, we have used a CYP1A post-translational inhibitor (FL) in order to calculate the contribution of CYP1A to the BFCOD assay. We also dosed with benzo[k]fluoranthene (BkF) and showed significant induction of CYP1A activity, with no concurrent increase in CYP3A activity. In this paper, we have taken an established in vitro CYP3A activity assay, and utilized the reaction in a novel way to allow for the non-destructive determination of CYP3A. In summary, we describe a sensitive, cheap, fast and easy modified BFCOD assay for in ovo and in vivo determination of CYP3A activity for use in moderate throughput early-life-stage fish experiments.     

Cytochrome P4501A induction in rainbow trout gills and liver following exposure to waterborne indigo, benzo[a]pyrene and 3,3',4,4',5-pentachlorobiphenyl

We have developed a gill-filament based ethoxyresorufin O-deethylase (EROD) assay to be used as a tool to monitor cytochrome P4501A (CYP1A) induction in caged fish. The present study aimed to compare temporal patterns of EROD induction in gills and liver of rainbow trout (Oncorhynchus mykiss) exposed in the laboratory to readily metabolized and persistent CYP1A inducers, i.e. indigo, benzo[a]pyrene (BaP), and 3,3',4,4',5-pentachlorobiphenyl (PCB#126). Branchial and hepatic EROD activities were examined in fish exposed for 6, 12, or 24h and in fish exposed for 24h and then held in clean water for 2 or 14 days. Furthermore, branchial CYP1A protein expression was localized by immunohistochemistry. All compounds strongly induced branchial EROD activity within 6h. The highest EROD inductions observed for indigo, BaP, and PCB#126 were roughly similar in gills (52-, 76-, and 74-fold), but differed considerably in liver (11-, 78-, and 200-fold). In indigo- and BaP-exposed fish, both hepatic and branchial EROD activities decreased rapidly in clean water. In PCB#126-exposed fish, decreased branchial and increased hepatic EROD activities were observed following transfer to clean water. The substances gave rise to immunostaining for CYP1A at different cellular sites. All inducers increased the CYP1A-immunostaining in the gill filament secondary lamellae, but PCB#126 also induced a pronounced CYP1A immunoreactivity in cells near the basal membrane of the epithelium of the primary lamellae. The observation that the low BaP and indigo concentrations induced EROD activity markedly in the gills but only slightly or not at all in the liver, supports the contention that readily metabolized AhR agonists may escape detection when hepatic EROD activity is used for environmental monitoring. The results show that gill filament EROD activity is a sensitive biomarker both for persistent and readily metabolized AhR agonists in polluted water.     

PCB77 (3,3',4,4'-tetrachlorobiphenyl) co-exposure prolongs CYP1A induction, and sustains oxidative stress in B(a)P-exposed turbot, Scophthalmus maximus, in a long-term study

Cytochrome P4501A (CYP1A), benzo(a)pyrene (B(a)P) activation and biliary elimination, phase II activities, and peroxisomal and antioxidant activities of turbot (Scophthalmus maximus) were studied in a long-term controlled experiment. Fish were serially exposed in water on day 1 and on completion of months 3, 6 and 9 to 0.1, 0.2, 0.1 and 0.1mg B(a)P/l, respectively, while another group was identically treated with additional PCB77 (3,3',4,4'-tetrachlorobiphenyl) at 1% of concomitant B(a)P (w/w). Temporally persistent responses were obtained by sampling on week 3 and 3 months from each latest exposure. Serial exposure to B(a)P+PCB77 progressively induced liver 7-ethoxyresorufin O-deethylase (EROD) activity and CYP1A protein levels (ELISA, western blotting) towards months 9, 12 and gill EROD activity on month 12. It associated with an apparent increase in liver benzo(a)pyrene diol epoxide (BPDE)-DNA adduct levels (ultrasensitive enzyme radioimmunoassay), and elevated bile B(a)P metabolite levels on month 9 females as compared to males. In contrast, B(a)P alone did not cause (p>0.05) comparable effects on liver EROD, CYP1A, adducts nor on bile metabolites. Both exposed groups demonstrated evidence for lasting oxidative stress as hepatic superoxide dismutase, catalase and glutathione peroxidase activities were significantly altered (p<0.05) with symptomatic pro-oxidant associations among them. Both treatments affected liver somatic index similarly (increase on month 3, decrease on month 9 in males). Continued exposure on month 18 (0.2mg B(a)P/l, 1% PCB77) followed by sampling 6 months later showed sustained induction (p<0.001) of hepatic EROD in B(a)P+PCB77 group, which was not seen in B(a)P alone treatment. Thus, PCB77 co-exposure prolonged CYP1A induction and contributed to a persistent oxidative challenge in B(a)P-exposed turbot. The results indicate synergistic effects of polycyclic aromatic hydrocarbon (PAH) and polychlorinated biphenyl (PCB) exposure in the aquatic environment.     

CYP1 and AhR expression in 7,12-dimethylbenz[a]anthracene-induced mammary carcinoma of rats prenatally exposed to 3,3',4,4',5-pentachlorobiphenyl

We previously reported the finding that prenatal exposure to a relatively low dose of 3,3',4,4',5-pentachlorobiphenyl (PCB126) acted as an enhancing agent for 17-beta-estradiol (E2)-dependent 7,12-dimethylbenz[a]anthracene (DMBA)-induced rat mammary carcinoma, while a high dose decreased it. E2 is a known risk factor for mammary carcinoma, and CYP1A1 and 1B1 (CYP1) are the major enzymes catalyzing 2- and 4-hydroxylation of E2, respectively. We investigated the induction of CYP1 and aryl hydrocarbon receptor (AhR) in DMBA-induced mammary carcinoma using female Sprague-Dawley rats whose dams had been treated (i.g.) with 2.5 ng, 250 ng, 7.5 microg of PCB126/kg or the vehicle on days 13-19 post-conception. Immunohistochemical analysis revealed that the mammary carcinoma of the 250 ng group showed a significantly higher number of nuclei expressing estrogen receptor alpha (ER) and proliferating cell nuclear antigen (PCNA) compared to those of the other groups. Quantitative real-time RT-PCR analysis revealed that the 7.5 microg group showed a significantly higher level of CYP1A1 mRNA, and that the 250 ng group showed significantly higher levels of CYP1B1 mRNA. The level of AhR mRNA was significantly higher in both the 7.5 microg and 250 ng groups. Western blotting analysis was consistent with mRNA changes. It has been revealed that CYP1B1 catalyzes a step in the formation of 4-hydroxylated E2 metabolites, which show quite high mammary carcinogenicity. This study indicates that the enhancement of DMBA-induced mammary carcinogenicity in a relatively low PCB126 dose group might partially involve the higher expression of CYP1B1 and AhR in these carcinomas.     

Relative contributions of affinity and intrinsic efficacy to aryl hydrocarbon receptor ligand potency

Models of receptor action are valuable for describing properties of ligand-receptor interactions and thereby contribute to mechanism-based risk assessment of receptor-mediated toxic effects. In order to build such a model for the aryl hydrocarbon receptor (AHR), binding affinities and CYP1A induction potencies were measured in PLHC-1 cells and were used to determine intrinsic efficacies for 10 halogenated aromatic hydrocarbons (HAH): 2,3,7, 8-tetrachlorodibenzo-p-dioxin (TCDD), 2,3,7, 8-tetrachlorodibenzofuran (TCDF), and eight polychlorinated biphenyls (PCB). TCDD, TCDF, and non-ortho-substituted PCBs 77, 81, 126, and 169 behaved as full agonists and displayed high-intrinsic efficacy. In contrast, the mono- and di-ortho-substituted PCBs bound to the AHR but displayed lower or no intrinsic efficacy. PCB 156 was a full agonist, but with an intrinsic efficacy 10- to 50-fold lower than non-ortho-substituted PCBs. PCB 118 was a very weak partial agonist. PCBs 105 and 128 were shown to be competitive antagonists in this system. The model was then used to predict CYP1A induction by binary mixtures. These predictions were tested with binary mixtures of PCB 126, 128, or 156 with TCDD. Both PCB 156 (a low-intrinsic efficacy agonist) and PCB 128 (a competitive antagonist) inhibited the response to TCDD, while the response to TCDD and PCB126 was additive. These data support the following conclusions: 1) only 1-2% of the receptors in the cell need be occupied to achieve 50% of maximal CYP1A induction by one of the high-intrinsic efficacy agonists, demonstrating the existence of "spare" receptors in this system; 2) the insensitivity of fish to ortho-substituted PCBs is due to both reduced affinity and reduced intrinsic efficacy compared to non-ortho-substituted PCBs; 3) PCB congeners exhibit distinct structure-affinity and structure-efficacy relationships. Separation of AHR ligand action into the properties of affinity and intrinsic efficacy allows for improved prediction of the behavior of complex mixtures of ligands, as well as mechanistic comparisons across species and toxic endpoints.