* Department of Pharmacology, University of Kentucky Medical Center, Lexington, Kentucky 40536; and
College of Pharmacy, University of New Mexico Health Sciences Center, Albuquerque, New Mexico 87131
Received September 9, 2000; accepted January 18, 2001
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ABSTRACT |
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Key Words: aryl hydrocarbon receptor; TCDD; cardiac malformations; chick embryo; halogenated aromatic hydrocarbons..
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INTRODUCTION |
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The aryl hydrocarbon receptor nuclear translocator (AHR) is a member of the basic helixloophelix/PAS (bHLH/PAS) protein family. Other members of this family include hypoxia-inducible factor (HIF-1), involved in mediating the physiological response to hypoxia (Wang et al., 1995
), a number of proteins that regulate circadian rhythms, such as Per, a protein involved in regulating neurogenesis, Sim (Muralidhar et al., 1993
), and coactivator proteins such as steroid receptor coactivator, Src-1 (Onate et al.,1995
). The evidence that the AHR, like many of the bHLH/PAS proteins, may play key roles during embryonic development includes the observations that 3 independent strains of AHR null mice display a number of irregularities, such as a slower growth rate, abnormal hepatic development, and impaired reproduction in females (Fernandez-Salguero et al., 1995
, 1997
; Mimura et al., 1997
; Schmidt et al., 1996
).
The AHR is a cytosolic receptor bound to a chaperone complex that contains two molecules of heat shock protein 90 and a novel immunophilin identified as ARA9 (AH receptor inhibitory protein) or AIP (AH receptor inhibitory protein) (Carver and Bradfield, 1997; Chen and Perdew, 1994
; Ma and Whitlock, 1997
). Upon binding of a ligand such as TCDD, the AHR translocates into the nucleus and dissociates from its chaperone complex in the nucleus (Heid et al., 2000
; Lees and Whitelaw, 1999
). The AHR subsequently dimerizes with its DNA binding partner, the AHR nuclear translocator (ARNT), and binds to specific DNA response elements known as dioxin-responsive elements (DRE), leading to an increase in transcription of certain genes, such as mammalian cytochrome P-450 1A1 and 1A2 (CYP1A1 and CYP1A2). However, the relationship between gene induction and the toxicity of compounds such as TCDD is not well understood (Hankinson, 1995
).
The AHR is the only characterized member of the bHLH/PAS protein family that is known to be ligand-activated. Currently, the best-characterized AHR ligands are xenobiotics such as TCDD. The chicken is one of the most sensitive species to TCDD-induced toxicity, which was first observed with the massive deaths of millions of broiler chickens in 1957 from "chick edema disease" after their feed was contaminated with trace amounts of TCDD and other HAHs (Higginbotham et al., 1968; Schmittle, et al., 1958
). Furthermore, the chick embryo has been used to characterize and study the carcinogenic and developmental effects of TCDD, and to elucidate many of the mechanisms involved in activation of the AHR, including structure/activity relationships between ligand activation of the AHR and CYP1A induction (Poland and Glover, 1973
1977). Two avian homologues to mammalian CYP1A1 and 1A2 that are induced by TCDD have been characterized and are designated as CYP1A4 and 1A5 (Rifkind et al., 1985
, 1994
).
One of the earliest and most sensitive developmental effects of TCDD in the chick embryo is cardiotoxicity, suggesting that the AHR may play a physiological role in heart development. This cardiotoxicity mediated by TCDD involves enlarged right and left ventricles, decreased responsiveness to ß-adrenergic agonists, abnormalities of conduction and heart contractility, and subcutaneous and pericardial edema, reflecting a progression to heart failure (Canga et al., 1988; Cheung et al., 1981
; 1993; Fan et al., 2000
; Walker and Catron, 2000
; Walker et al., 1997
). When injected into the yolk of chicken eggs prior to incubation, TCDD induces a dose-related increase in heart wet weight on incubation-day 10 that is associated with an increase in cardiac myosin content (Walker and Catron, 2000
). Since the increase in day-10 heart wet weight induced by TCDD is indicative of cardiotoxicity during chick embryo development, it may be used as an endpoint to assess the cardiotoxic potency of other HAH congeners in vivo.
Cardiotoxic effects have been observed in other species in response to TCDD exposure and AHR activation as well. TCDD exposure resulted in pericardial edema in zebrafish (Danio rerio; Henry et al., 1997) and reduction in heart size in rainbow trout (Oncorhynchus mykiss; Hornung et al., 1999). Recent studies using AHR-null mice have lead to the discovery of cardiac hypertrophy in adults (Fernandez-Salguero et al., 1997) and in developing embryos (Thackaberry and Smith et al., 1999), which implicates a role for the AHR in the developing murine heart. The expression of the AHR and ARNT in the chick embryo has been localized to regions within the heart that are susceptible to TCDD-induced cardiotoxicity, and thus supports the idea that the AHR may mediate these TCDD-induced effects (Walker and Catron, 2000
; Walker et al., 1997
). However, no studies have established a direct correlation between AHR activation and cardiotoxicity in the chick embryo. Therefore, our objective was to determine whether a correlative relationship exists between AHR activation and subsequent cardiotoxicity by HAHs, thereby providing a model for further study of the physiological function of the AHR in cardiac development.
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MATERIALS AND METHODS |
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Cardiotoxicity of HAH congeners in vivo.
Fertile Plymouth Rock, barred variety chicken eggs were obtained from Privett Hatchery (Portales, NM) and stored for less than 5 days at 14°C until used for egg injection experiments. Initial range-finding studies were conducted with each congener prior to final dose-response experiments and a minimum of 3 dose-response experiments were conducted with each congener. In all cases, eggs were injected into the yolk prior to incubation with 5 µl corn oil (vehicle), 4 graded doses of TCDD dissolved in 5 µl of corn oil, 4 graded doses of another HAH congener, or a single dose of HCB 153 dissolved in 5 µl corn oil; eggs were sealed with paraffin wax and incubated for 10 days (D10) at 37.5°C, 55% humidity. Finally, dose-response studies were conducted with the following doses: 0.06, 0.12, 0.24, 0.40, or 0.8 pmol TCDD/g (19258 pg/g); 0.1, 0.2, 0.4, or 0.8 pmol PCDD/g (36285 pg/g); 0.12, 0.24, 0.36, or 0.48 pmol TCDF/g (37122 pg/g); 0.75, 1.5, 2.0, or 3.0 pmol PCDF/g (2561,023 pg/g); 2, 4, 6, or 8 pmol PCB 77(3,3',4,4'-tetrachlorobiphenyl)/g (5842336 pg/g); or a single dose of 10,000 pmol PCB 153/g (3610 ng/g). Twelve eggs were injected for the control, the 3 lowest doses of each congener, and a single dose of PCB 153, while 18 eggs were injected for the highest dose of each congener. On D10, hearts were dissected from each embryo, rinsed in ice-cold PBS, blotted on waxed paper, and wet weights determined.
Oligonucleotides.
Oligonucleotides were purchased from Gibco BRL (Gaithersburg, MD). The annealed oligonucleotides that were used as the radiolabeled probe and the wild-type competitor for the electrophoretic mobility shift assay (EMSAs) and contain the DRE (underlined) are: 5'-TCGAGCTGGGGGCATTGCGTGACATTAC (HIS 17) and 3'-TCGAGGTATGTCACGCAATGCCCCCAGC (HIS 18). The lower sequence had been determined previously to be the optimal DNA recognition site of the AHR and ARNT complex (Swanson et al., 1995). The annealed nucleotides that were used as the competitor oligonucleotides for the EMSAs containing the mutated DRE (underlined) are: 5'-TCGAGCTGGGGGCATTGATTGACATAC (HIS 108) and 3'-TCGAGGTATGTCAATCAATGCCCCCAGC (HIS 109).
Plasmids and antibodies.
The plasmid that was used to generate the stably transfected HepG2 cell line for the luciferase assays, pLUC1A1, was provided by Dr. Robert Tukey (UC San Diego). The HepG2-p450luc cell line was generated following transfection of HepG2 cells with the pLUC1A1 plasmid and clonal selection using neomycin. The luciferase reporter vector (pGL3/DRE2) used for the transient transfection of LMH cells contains 2 copies of the DRE sequence (HIS 17/18) inserted into the Xho I site of pGL3 (Promega). The anti-AHR immunoglobulins used for all experiments and the anti-ARNT immunoglobulins used for the EMSO supershift experiments were described previously (Pollenz et al., 1994), and were a generous gift from Dr. Richard Pollenz (Charleston, SC). The cytochrome p450 1A1 antibody was purchased from XenoTech, LLC Kansas City, KS and the actin antibody from Sigma.
Reagents.
PBS (phosphate-buffered saline) contains 150 mM NaCl, 3 mM KCl, 1.5 mM KH2PO4, and 10 mM Na2HPO4, pH 7.4. TTBS contains 50 mM Tris, 0.2% Tween 20 and 150 mM NaCl, pH 7.5. TTBS+ contains 50 mM Tris, 0.5% Tween 20, and 300 mM NaCl, pH 7.5. BLOTTO contains 5% nonfat dry milk in TTBS. MENG is 25 mM MOPS pH 7.5, 1 mM EDTA, 0.02% sodium azide, 10% glycerol. Lysis buffer is 100 mM K2HPO4 and 1 mM DTT. Ligand lysis buffer is 25 mM Tris, 15% glycerol (v/v), 2% CHAPS, 1% lecithin, 1% BSA (w/v), 4 mM EGTA, 1 mM DTT, 0.4 mM PMSF, and 8 mM MgCl2. F buffer is 10 mM Tris, pH 7.05, 50 mM NaCl, 30 mM sodium pyrophosphate, 50 mM NaF, 5 µM ZnCl2, 0.1 mM Na3VO4, 1% Triton x-100, 1 mM PMSF, 1 µg/ml 2-macroglobulin, 1 µg/ml pepstatin A, 1 µg/ml leupeptin, 150 µM benzamidine, 2.8 µg/ml aprotinin. HDK is 25 mM HEPES, 1 mM DTT, 0.4 M KCl. MDHK is 3 mM MgCl2, 1 mM DTT, 25 mM HEPES, 0.1 M KCl. MDH is 3 mM MgCl2, 25 mM HEPES, 1 mM DTT. TBE is 45-mM Tris base, 45-mM boric acid, and 1-mM EDTA, pH 8.0.
Tissue culture.
HepG2 cells (a human hepatoma cell line) were cultured until nearly confluent in Dulbecco's modified eagle medium and 8% fetal bovine serum. LMH cells (a chicken hepatoma cell line) were obtained from ATCC (Manassas, Va) and were cultured in Waymouth MB 752/1 medium and 8% fetal bovine serum as described previously (Kawaguchi et al., 1987). The cells were treated with the indicated concentrations of agonist for 1 h prior to isolation of nuclear extracts or for 16 h prior to harvesting for analysis by Western blot or luciferase activity. Protein concentrations were determined by the BCA protein assay (Pierce).
Western blot analysis.
HepG2 cells were treated with the indicated concentration of agonist, and whole cell lysates were isolated as described previously (Sommer et al., 1998). Eighty micrograms of each sample were analyzed by SDSPAGE and Western blot analysis as described previously (Heid et al., 2000
). The primary antibodies, anti-cytochrome p4501A1 and anti-actin, were diluted in BLOTTO and used for immunostaining (1:1,000 and 1:500, respectively). The secondary antibody was linked to horseradish peroxidase (HRP; Pierce; 1:10,000) and the specific protein bands were visualized using the ECL detection system (Amersham).
EMSA analysis of nuclear extracts.
Nuclear extracts were prepared from HepG2 or LMH cells as described previously (Heid et al., 2000). Aliquots of the nuclear extracts (6 µg) were incubated with 4 µg salmon testes DNA and KCl (0.1 M final concentration) in MENG buffer at room temperature for 10 min. The samples were then incubated with 32P-labeled HIS 17/18 for 10 min at room temperature, and an additional 15 min with a specific antibody for supershifting, where indicated. The samples were applied to a non-denaturing 4% poly-acrylamide gel using 0.5 x TBE as the running buffer. The results were then quantitated using phosphorimaging analysis.
Luciferase assays.
The treated HepG2-p450luc cells were washed twice with PBS and harvested into lysis buffer. The cells were then lysed by 3 cycles of freeze/thaw. Next, the samples were centrifuged for 10 min at 8000 rpm at 4°C, and the supernatants were analyzed by the luciferase assay. The luciferase analysis was performed as outlined by Promega (Madison, WI). Results were normalized by protein concentration as determined by the BCA protein assay (Pierce).
LMH cells were grown until approximately 5060% confluent and transiently transfected in 100 mM dishes with 5 µg of pGL3/DRE2 (containing two DREs upstream of a luciferase reporter gene), 5 µg of pRSV/ß-gal (containing a ß-galactosidase gene for normalization), and 10 µg pUC19 (carrier DNA), using the calcium phosphate method as described previously (Swanson and Yang 1996). After 34 h, the cells were treated with the indicated concentrations of congeners and incubated an additional 16 h. The cells were lysed in ligand lysis buffer and luciferase activity analyzed as described above. The luciferase values were normalized by ß-galactosidase concentration as determined by the ß-galactosidase assay (Promega, Madison, WI).
Statistical analysis.
A continuous dose-response curve was generated for heart wet weight as a function of HAH dose, using a probit procedure. Each congener, except PCB 153, exhibited full efficacy (as compared to TCDD) in increasing heart wet weight, with an average maximal increase of 29.9 ± 2.9% above control. Thus, the effective dose, which increased heart wet weight above control by 15%, was defined as the ED50 and was used to calculate RP (relative potency) values. The RP values were calculated by dividing the TCDD ED50 by the HAH congener ED50. The reported RP values for cardiotoxicity are based on 3 independent experiments. To determine the EC50 values from the dose-response data for each congener in the in vitro studies, the phosphorimaging data for each EMSA were analyzed by the PRISM (San Diego, CA) statistical analysis program. The RP values were calculated by dividing the TCDD EC50 by that obtained for each congener and are representative of 3 independent experiments. Pearson product-moment correlation analysis was used to determine statistical significance for correlation of the RP values obtained from the heart wet weight increase and EMSA analyses. One-way and 2-way ANOVA tests were used to determine statistical significance among the dose-response data of the differing agonists. Where applicable, Tukey's pairwise comparison test was also used to analyze the dose-response data between differing agonists. As a means of determining statistical outliers, we used the studentized residual test provided by SigmaStat software.
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RESULTS |
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Induction of cardiotoxicity by TCDD and other HAH congeners in the chick embryo.
To establish an effective dose for chick embryo cardiotoxicity, fertile chicken eggs were injected with vehicle, graded doses of TCDD, or other HAH congeners prior to incubation and heart wet weights measured on D-10. This protocol had been used previously to demonstrate a dose-related increase in D10 heart wet weight that appropriately reflected the cardiotoxic actions of TCDD (Walker and Catron, 2000). A typical dose-response curve that was generated by TCDD or PCB 77 treatment is shown in Figure 1
.
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Activation of AHR/ARNT/DNA binding by other HAH congeners.
The 5 remaining HAH congeners were analyzed in a similar manner as TCDD in both LMH and HepG2 cells and EC50s for DNA binding were calculated (Table 1). The rank/order for the EC50's was significantly correlated between the 2 cell lines (r = 0.95, p = 0.05). TCDD and PCDF were the most potent in activating the formation of the AHR/ARNT/DNA-binding complex, whereas TCDF and PCDD exhibited intermediate potencies, PCB 77 exhibited low potency, and PCB 153 failed to activate an AHR/ARNT/DNA binding complex above that observed in the DMSO control.
Comparison of cardiotoxicity induction and AHR activation by HAHs.
The correlation between each congener's ability to induce cardiotoxicity and to activate DNA binding of the AHR was determined by comparing the respective relative potencies (Table 1). When comparing the ability of these compounds to induce formation of the AHR/ARNT/DNA binding complex in chicken LMH cells to their ability to induce cardiotoxicity in the chick embryo, the rank/order for 5 of the compounds (TCDD, PCDD, TCDF, PCB 77 and PCB 153) produced a significant positive correlation (r = 0.94, p = 0.017). In contrast, the RP of PCDF in inducing cardiotoxicity and AHR activation differed by approximately 10-fold (cardiotoxicity RP = 0.11 as compared to AHR/ARNT DNA binding RP = 0.95, Table 1
). When the RP values for PCDF were included in the correlation analysis, the correlation coefficient dramatically changed (r = 0.53, p = 0.28). The studentized residual test was used to determine whether the PCDF data point could be considered as a statistical outlier. Given that the PCDF value was outside of the 95% confidence interval for the best-fit regression line between the data points, we considered the PCDF value to be an outlier.
Gene induction by HAHs using luciferase reporter assays.
To verify that the analysis of AHR/ARNT/DNA binding shown in Figure 2 can accurately predict the activation of the AHR signaling pathway, we chose to examine the ability of HAHs to induce the expression of AHR-regulated genes. Towards this end, we analyzed the ability of TCDD and the less-potent PCB 77 and PCDF congeners to induce AHR gene activation of a DRE-regulated luciferase construct in both HepG2 and LMH cells. We first used the HepG2-P-450luc cell line, which is stably transfected with a luciferase reporter gene regulated by the promoter region of cytochrome P-4501A1. Administration of the respective agonists in this model revealed that the rank/order potencies for TCDD, PCB 77, and PCDF mirrored the values obtained by the EMSA analysis (Figs. 3A and 3B
). These results also confirm that analysis of DNA binding of the AHR/ARNT complex is a reliable indicator of AHR activation.
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Induction of cytochrome P-450 1A1 by HAHs.
As an additional endpoint for estimating the ability of TCDD and PCDF to induce the AHR signaling pathway, we analyzed induction of CYP1A1 in HepG2 cells. Here, treatment with TCDD and PCDF at similar concentrations induced relatively equal amounts of CYP1A1 protein levels in HepG2 cells (Fig. 4). Thus, TCDD and PCDF may be considered approximately equipotent activators of the AHR signaling pathway in cultured hepatoma cells.
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DISCUSSION |
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Potencies of HAH congeners for chick embryo cardiotoxicity.
All HAH congeners tested, except PCB 153, were able to induce dose-related increases in heart wet weight on day 10. The rank order potency for this in vivo toxicity was TCDD > PCDD = TCDF > PCDF > PCB 77; PCB 153 no effect. Although the toxic potency of most of these HAHs have not been previously studied in an avian model, the relative potency of PCB 77 for inducing cardiotoxicity in this study (0.03) is similar to that reported for inducing thymic involution in chick embryos (Nikolaidis et al., 1988; Rifkind et al., 1985
).
However, the rank order of toxic potency of these 6 HAH congeners in the chick embryo differs from that causing toxicity in fish (Walker and Peterson, 1991). In fish, the rank order potency of these 6 HAH congeners for causing rainbow trout early life-stage mortality is: TCDD > PCDD > PCDF > TCDF > PCB 77, PCB 153 no effect (Walker and Peterson, 1991
). When comparing the rank order of toxic potency, TCDF is ranked higher for causing chick embryo cardiotoxicity than its ranking for inducing fish early life-stage mortality. For example, the RP for the TCDF-induced increase in heart wet weight in chick embryos is 0.55 compared to the RP of 0.028 for inducing fish early life-stage mortality. In addition, PCB 77 exhibits the lowest potency of this group of congeners in both model systems, but is significantly more potent during chick embryo development than fish development. The RP for PCB 77 based on chick cardiotoxicity is 0.03, compared to the RP of 0.00016 for fish developmental mortality.
The rank order toxic potency of these 6 HAH congeners in the chick embryo also differs from the rank order toxic potency of these HAHs proposed for human health-risk assessment (Safe, 1990). For example, the RP of TCDF for inducing chick embryo cardiotoxicity is 0.55, while the toxic equivalency factor (TEF) that is derived from multiple studies and is proposed for human health-risk assessment is 0.1 (Safe, 1990
). In addition, PCB 77 is approximately 3 times more potent in causing chick embryo cardiotoxicity than mammalian toxicity. The values obtained in this study support the idea that the application of TEFs from human health-risk assessment to avian wildlife risk assessment may underestimate the total risk posed by environmental HAH mixtures, particularly when TCDF and/or PCB 77 represent a large component of that mixture (Van den Berg et al., 1998
).
It is not known what accounts for the differences in the structure-activity relationships for these HAHs among birds, fish, and mammals. Studies in mammals have shown that their AHR binding affinity is highly correlated with their ability to induce expression of CYP1A1 and their in vivo toxic potency (Bandiera et al., 1984; Mason et al., 1986
; Safe et al., 1985
). Thus, differences in the structure-activity relationships observed among birds, fish, and mammals may result from differences in the ligand-binding domain of the AHR. This idea is supported by the finding that TCDF and PCB 77 are 34 times more potent in inducing DNA binding of the AHR/ARNT complex in the chicken LMH cells when compared to that in the human HepG2 cells (Table 1
). Since the AHR has been cloned from fish (Abnet et al., 1999
; Hahn et al., 1997
; Roy and Wirgin, 1997
; Tanguay et al., 1999
), birds (Karchner et al., 2000
; Walker et al., 2000
), and mammals, the contribution of AHR binding affinity to differences in structure-activity relationships among phyla can now be assessed.
Comparison of HAH potencies between in vivo toxicity and in vitro DNA binding.
Many advantages exist for using cell culture systems to estimate the toxic potency of individual HAHs as well as the combined toxic potency of complex environmental mixtures. These assays systems are inexpensive, rapid, and allow for many samples to be tested simultaneously. One significant shortcoming to the use of these cell culture assay systems is the failure to validate whether the results from cell culture accurately predict the toxic potency of HAHs in vivo. Thus, one of our primary objectives was to determine whether the potency of individual HAH congeners assessed in vitro would correlate with their toxic potency in vivo.
We found a significant correlation between in vitro AHR/ARNT/DNA binding in a chicken LMH hepatocyte cell line and in vivo cardiotoxicity for 5 HAH congeners: TCDD, PCDD, TCDF, PCB 77, and PCB 153. In these model systems, TCDD was the most potent in causing the 2 endpoints measured, respectively, while PCB 153 had no effect in either system. Although PCDD, TCDF, and PCB 77 did not exhibit identical potencies in the 2 systems, their relative potencies were highly correlated. The potency of one congener, PCDF, in the 2 model systems failed to fit the correlation exhibited by the other 5 HAH congeners. PCDF was nearly 10-fold more potent in inducing DNA binding of the AHR/ARNT complex in the LMH cell line than in inducing cardiotoxicity in the chick embryo (Table 1). The observation that PCDF exhibits significantly weaker potency in inducing cardiotoxicity than in activating the AHR signaling pathway indicates that PCDF is an outlier in the RP correlations. The explanation for this difference in potency is not known; however, differences in its distribution profile may contribute to this difference. An alternative explanation for PCDF's contrasting potencies in these 2 models could involve its interaction with an additional receptor or protein that alters its effect on cardiac development. However, the fact that PCDF induces a significant increase in heart wet weights still supports the idea that AHR activation mediates the in vivo cardiotoxicity.
One of the problems associated with the use of RPs established in vitro to predict whole-animal risk is whether the endpoint selected to assess potency has physiological relevance (Sutter, 1995). With this in mind, we also chose to measure endpoints that occur downstream of AHR/ARNT/DNA binding, including the induction of gene activation using reporter constructs and induction of endogenous protein expression for CYP1A1 for a select number of HAH congeners. Both of these endpoints confirmed the RP data generated using AHR/ARNT/DNA binding as an endpoint; the potency of PCDF in vitro failed to correlate with its toxic potency in vivo, while the potency of TCDD and PCB 77 remained highly correlated with in vivo cardiotoxicity.
Potential mechanism of HAH-induced cardiotoxicity.
The strong correlation between the ability of HAH congeners to activate the AHR signaling pathway and produce cardiotoxicity during chick embryo development suggests that this toxic response may be mediated by the AHR. AHR agonists may induce the cardiotoxic response by a number of potential mechanisms. For example, the HAH may activate the AHR at an inappropriate time or to an inappropriate level during embryonic cardiomorphogenesis. This activation could sequester ARNT from its role in mediating the response of HIF-1, a protein involved in coronary angiogenesis, thereby resulting in poor myocardial vascularization and subsequent cardiac dilation. The potential for an activated AHR to participate in cross talk with the ARNT/HIF-1
signaling pathway has been demonstrated previously under certain experimental conditions (Chan et al., 1999
; Gradin et al., 1996
); however, the ability of this to occur in vivo has not been explored. Another potential mechanism of cardiotoxicity by AHR ligands may simply be the direct or indirect activation and/or suppression of genes that regulate developmental cardiac morphogenesis. The identification of these cardiac genes is not currently known; however, preliminary data suggest that the AHR agonist TCDD down-regulates expression of the cardiac Na+/K+ ATPase alpha subunit (Walker, 1999
), which could contribute to an alteration in cardiac contractility (Fan et al., 2000
) and ventricle cavity dilation (Walker and Catron, 2000
).
Conclusions.
These results indicate that in general, the ability of HAH congeners to activate the AHR signaling pathway correlates with their ability to mediate cardiotoxicity in the chick embryo. This observation, together with recent studies that have detected expression of the AHR and ARNT in regions of the hearts that are responsive to TCDD's toxic effects (Walker et al., 1997, 2000
), provide strong evidence for a role for the AHR in mediating cardiotoxicity by HAHs. Furthermore, these results, together with the observations that AHR null mice exhibit cardiac hypertrophy during embryo development (Thackaberry and Smith, 1999
) and adulthood (Fernandez-Salguero et al., 1997
), imply a potential physiological function for the AHR in cardiac development and growth.
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ACKNOWLEDGMENTS |
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NOTES |
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