![[Feedback]](/icons/banner/feedbackACT.gif){kind=icon}
![[For Subscribers]](/icons/banner/subscriberACT.gif){kind=icon}
![[Archive]](/icons/banner/archiveACT.gif){kind=icon}
![[Search]](/icons/banner/searchACT.gif){kind=icon}
![[Contents]](/icons/banner/tocACT.gif){kind=icon}
|
|
|
|
|
||
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Iconix Biosciences, Mountain View, California (W.H., M.R.F.); Department of Environmental Toxicology, University of California Davis, Davis, California (C.S., M.S.D.); and Roche Palo Alto LCC, Palo Alto, California (K.K.)
Received November 15, 2006; accepted February 27, 2007
 |
Abstract |
|---|
 Top Abstract Materials and MethodsResultsDiscussionReferences |
|---|
).
The induction of Cyp1a1 mRNA and resulting enzyme activity has long been used as a sensitive indicator of AhR activation in numerous in vitro and in vivo models to screen a variety of compounds, mixtures, and environmental matrices (Behnisch et al., 2001). As a result of the strong correlation observed between AhR binding affinity, Cyp1a1 induction, and dioxin-like toxicity of structurally related HAHs, Cyp1a1 induction has been used as a biomarker for hazard identification and risk assessment of environmental pollutants, industrial chemicals, and therapeutic compounds (Behnisch et al., 2001, 2002). Such use assumes that induction of Cyp1a1 is specifically associated with AhR activation and that activation of the AhR leads to dioxin-like toxicity. In contrast to this assumption, AhR-independent induction of Cyp1a1 has been documented (Delesculuse et al., 2000), and nonhalogenated high-affinity ligands of the AhR such as 
-naphthoflavone or high doses of weaker or labile endogenous ligands such as prostaglandins (Seidel et al., 2001), heme degradation products (Phelan et al., 1998), and tryptophan metabolites (Heath-Pagliuso et al., 1998) fail to induce dioxin-like toxicities in rodents. In addition, the AhR has been shown to bind and be activated by a diverse range of chemicals whose structures are dramatically different from the typical planar hydrophobic AhR agonists (Denison et al., 1998, 2002; Denison and Nagy, 2003). These findings raise questions about the validity of the use of Cyp1a1 and related enzyme activity as a specific biomarker of AhR activation and the relevancy of HAH-induced effects to the safety assessment of nonpersistent AhR agonists.
To evaluate the accuracy of in vivo Cyp1a1 induction as a biomarker of AhR agonist activity, we evaluated rat gene expression data in DrugMatrix, a large toxicogenomic database of gene expression profiles for 596 compounds (Ganter et al., 2005), and found that Cyp1a1 was induced by 239 compounds in a variety of tissues. The majority of the active compounds are marketed drugs with toxicity profiles unlike those produced by exposure to HAHs. To evaluate the sensitivity and specificity of in vivo Cyp1a1 induction to identify AhR agonists, a subset of 147 compounds was evaluated using a combination of in vitro assays to assess their ability to stimulate AhR transformation and DNA binding, dioxin response element (DRE)-driven reporter gene expression, and to compete with dioxin for binding to the AhR. The in vivo expression of other AhR-regulated genes, including Cyp1a2, Ugt1a1, and Nqo1, was also evaluated to determine whether the expression of these DRE-driven genes could improve the accuracy for identifying AhR agonists. Although all AhR agonists induce Cyp1a1 gene expression, the induction of Cyp1a1 expression in vivo does not necessarily implicate that a chemical is a direct AhR agonist. Furthermore, six marketed drugs that activate and bind to the rat AhR were identified and many treatments that induce Cyp1a1 in a tissue-specific manner and in a distinct pattern relative to other AhR-regulated genes. These results lend support to the hypothesis that AhR activation is not synonymous with AhR agonist activity and HAH-like toxicity for nonpersistent compounds.
|
Materials and Methods |
|---|
|
TopAbstractMaterials and MethodsResultsDiscussionReferences |
|---|
). This includes data on 596 compounds representing 3230 compound-dose-time point combinations. In brief, in vivo short-term repeat-dose rat studies have been conducted previously by Iconix Biosciences on reference compounds, including marketed, discontinued, and withdrawn drugs and toxicological and biochemical standards. For each compound, 6- to 8-week-old male Sprague-Dawley rats [Crl:CD (SD)(IGS)BR; Charles River Laboratories, Portage, MI] (three per group) were dosed daily at either a low (fully effective) or high (maximum tolerated) dose intended to reduce body weight gain or induce histopathological tissue injury. Animals were necropsied on days 0.25, 1, 3, and 5 or 7. Liver, kidney, or heart tissues from treated rats were profiled for gene expression in biological triplicate on the CodeLink RU1 microarray platform (GE Healthcare, Chalfont St. Giles, Buckinghamshire, UK). Housing and treatment of the animals were in accordance with regulations outlined in the United States Department of Agriculture Animal Welfare Act (9 CFR parts 1, 2, and 3)
Gene Expression Profiling. Gene expression profiling, data processing, quality control, and statistical analysis were performed as described previously (Ganter et al., 2005). The microarray gene expression results reported herein are presented as log10 ratios for Cyp1a1 (GenBank accession no. X00469
[GenBank]
), Cyp1a2 (GenBank accession no. K02422
[GenBank]
), Ugt1a1 (GenBank accession no. J05132
[GenBank]
), and Nqo1 (GenBank accession no. NM_017000
[GenBank]
), in which each experimental group is computed as the difference between the average of the logs of the normalized experimental signals and the average of the logs of the normalized control signals for each gene. Treatment-related effects on gene expression were considered significant at p < 0.05. All gene expression data presented herein for all 596 compounds, representing 3230 compound-dose-time point combinations in liver, kidney, and heart, are provided in Supplementary Table S1.
Compound Selection for in Vitro AhR Screening. The 147 compounds analyzed in vitro were selected based on in vivo gene expression data to represent a diverse set of compounds that either induce, repress, or do not significantly affect Cyp1a1 transcript levels in the liver, kidney, or heart of treated rats. A number of these compounds that do not significantly induce Cyp1a1 in vivo were chosen to evaluate the potential for false negatives in the gene expression data. The compounds were obtained from a variety of different sources as described previously (Ganter et al., 2005). A summary of all in vitro data presented herein for all 147 compounds is provided in Table 1.
|
Electrophoretic Mobility Shift Assay. All 147 compounds were tested for their ability to transform the rat hepatic cytosolic AhR into its DNA binding form using electrophoretic mobility shift assay (EMSA) as described previously in detail (Denison et al., 2002). In brief, hepatic cytosol, prepared from 150-g male Sprague-Dawley rats (Charles River Laboratories) in HEDG buffer [25 mM HEPES, pH 7.4, 1 mM EDTA, 1 mM dithiothreitol, and 10% (v/v) glycerol] (Denison et al., 2002), was incubated with dimethyl sulfoxide (DMSO) (10 µl/ml), TCDD (20 nM), or the indicated test compound (10 µM) for 2 h at room temperature. Ligand-dependent AhR binding to a 
32P-labeled oligonucleotide probe containing the mouse DRE3 sequence (5'-GATCTGGCTCTTCTCACGCAACTCCG-3' and 5'-GATCCGGAGTTGCGTGAGAAGAGCCA-3') was resolved by nondenaturing polyacrylamide gel electrophoresis, and the amount of inducible protein/[32P]DNA complex formation was determined by PhosphorImager analysis (GE Healthcare). The difference between the amount of radioactivity in the induced protein/DNA complex in a treated sample lane minus that present in the identical position in a vehicle control (DMSO) lane represented the amount of specific protein/DNA complex, and the results were expressed as a percentage of the amount of protein/DNA complex induced by 20 nM TCDD. The assay was performed six times for each test compound, and a compound was considered positive if it produced a visible band on the gel in at least three of the six replicate experiments.
Luciferase Reporter Gene Assay. All 147 compounds were evaluated for their ability to induce AhR-mediated DRE-driven reporter gene expression using a recombinant H4IIE 1.1 rat hepatoma (H4L1.1c4) cell line stably transfected with DRE-driven firefly luciferase reporter gene directly under inducible control of the AhR. The cells were generated, grown, and maintained as described previously (Garrison et al., 1996). DMSO (10 µl/ml), TCDD (1 nM), or test compound (10 µM) was added to the 96-well culture plate containing a monolayer of cells. After 4 h of incubation at 37°C, the cells were lysed, and luciferase activity in an aliquot (50 µl) was determined using an Anthos Lucy 2 microplate luminometer. Each compound was tested in triplicate in three independent experiments, and the results were expressed as a percentage of the luciferase activity induced by 1 nM TCDD. Statistical significance of the differences in luciferase activities between treatments and vehicle controls was determined with a Student's t test (p < 0.01). In addition, only increases in luciferase activity greater than 10% of 1 nM TCDD were considered biologically relevant.
Ah Receptor-Ligand Binding Assay. To confirm the ability of a compound to directly bind to the AhR, a competitive ligand binding assay was performed on compounds positive in both the reporter gene assay and the gel-shift assay using methods detailed elsewhere (Denison et al., 2002) with minor modifications. In brief, 500-µl aliquots of a rat cytosolic preparation (2 mg/ml total protein concentration) were preincubated at room temperature for 30 min with the compound of interest (10 µM), TCDF (200 nM), or with an equal volume of DMSO. [3H]TCDD was then added to a final concentration of 20 nM. After 2 h, 200-µl aliquots of the incubation mixture were added to tubes containing 250 µl of HAP (0.5 mg/µl in HEDG buffer) and allowed to incubate for 30 min. Samples were centrifuged, and pellets were washed three times with HEDG buffer containing 0.05% (v/v) Tween 80. The radioactivity remaining in the HAP pellet was determined by liquid scintillation counting. Specific [3H]TCDD binding was determined by subtracting the radioactivity measured in the TCDF samples (nonspecific binding) from that measured in the samples that were incubated with [3H]TCDD alone (total binding). The assay was performed in triplicate for each compound, and the results are presented as a mean percentage of the displacement of specific [3H]TCDD binding.
|
Results |
|---|
|
TopAbstractMaterials and MethodsResultsDiscussionReferences |
|---|
-naphthoflavone (BNF; 1500 mg/kg/day) and 3-methylcholanthrene (3-MC; 300 mg/kg/day) significantly induced Cyp1a1, Cyp1a2, Ugt1a1, and Nqo1 in the liver at multiple time points, although the results for 3-MC were more variable for Ugt1a1 and Nqo1 (Fig. 1A). In addition, there were many treatments, including albendazole, hydralazine, leflunomide, omeprazole, and others that caused similar significant changes in gene expression across these AhR-regulated and 3-MC/BNF-inducible genes, suggesting that these compounds are potential AhR agonists (Fig. 1B). Other than omeprazole (Shih et al., 1999), these compounds have not been described previously as Cyp1a1 inducers or as AhR agonists. Cyp1a1 was induced more than 100-fold by leflunomide and phenothiazine. Consistent with previous findings, the benzimidazole drugs lansoprazole and rabeprazole had strong effects (>100-fold) on Cyp1a1 (Backlund et al., 1999). Omeprazole also induced Cyp1a1 90-fold, which is consistent with published findings showing induction of Cyp1a1 in hepatocytes from a number of species (Shih et al., 1999). By comparison, BNF and 3-MC maximally induced Cyp1a1 61- and 7-fold, respectively. It is interesting that the pineal gland hormone melatonin significantly induced Cyp1a1 more than 32-fold in addition to inducing Cyp1a2, Ugt1a1, and Nqo1 (Fig. 1B).
|
Coregulation of Cyp1a1 and Other AhR-Regulated Genes. There were a large number of treatments that significantly induced Cyp1a1 but not Cyp1a2, Ugt1a1, and Nqo1 concurrently. This included 73 treatments in liver, 134 in heart, and 75 in kidney (Fig. 2 and Supplementary Table S1). Many of these treatments slightly but not significantly increased the levels of these other AhR-regulated genes, thus suggesting a weak AhR agonist effect. However, there were a number of compounds that clearly had no effect on these genes or even repressed them yet significantly induced Cyp1a1 (Fig. 2). In liver, for example, a number of toxicants such as 1-naphthyl isothiocyanate, ethanol, N-nitrosodiethylamine, and valproic acid significantly induced Cyp1a1 but slightly repressed Cyp1a2 at both early and late time points (Fig. 2A). A similar effect was particularly evident in heart, in which a number of compounds significantly induced Cyp1a1 but significantly repressed Cyp1a2, including bromisovalum (days 1 and 5), clofibric acid (days 3 and 5), isoprenaline (days 1 and 5), and vinorelbine (day 3) (Fig. 2B). Similar effects in kidney were observed for bromisovalum, cadmium acetate, and rifampin, although repression of Cyp1a2 was not as pronounced (Fig. 2C). Dexfenfluramine, whose metabolite is a potent 5-HT2B receptor agonist, also significantly induced Cyp1a1 in heart (Fig. 2B), but unlike the 5-HT2B receptor agonist BW-723C86, it did not induce Cyp1a2. This effect in heart was not evident in kidney, in which both Cyp1a1 and 1a2 were not significantly affected by dexfenfluramine (Supplementary Table S1). These results indicate that Cyp1a1 may not be coregulated with other AhR-regulated genes in heart and kidney. Furthermore, it suggests that Cyp1a1 is under regulatory control mechanisms distinct from the classic ligand binding and DRE-mediated transcription through the AhR or that tissue-specific factors are needed to support the induction of other DRE-regulated genes in these tissues.
|
Tissue-Specific Induction of Cyp1a1. Because of the disparate induction pattern of Cyp1a1 compared with other AhR-responsive genes under certain treatment conditions, it was of interest to determine whether similar effects on Cyp1a1 were observed across tissues. Of the 207 compound-dose-time point combinations that were profiled in more than one tissue and significantly induced Cyp1a1 in at least one of those tissues, only 41 (20%) did so in 2 of the 3 tissues examined. For example, none of the 64 compound-dose-time point combinations that were profiled in all 3 tissues significantly induced Cyp1a1 consistently across all 3 tissues (Fig. 3A). It is interesting that kidney-specific induction of Cyp1a1 was observed with the class of HMG-CoA reductase inhibitors. Consistent with the effects of mevastatin and lovastatin in kidney (Fig. 1D), other HMG-CoA reductase inhibitors, including cerivastatin, atorvastatin, pravastatin, and simvastatin significantly induced Cyp1a1 in kidney but not liver (Fig. 3B). The exception was cerivastatin on day 5, which significantly induced liver Cyp1a1 just more than 2.5-fold. These results indicate that induction of Cyp1a1 can be tissue-specific depending on the inducing agent.
|
Sensitivity of in Vivo Cyp1a1 Induction for Identifying AhR Agonists. To determine whether the observed induction of Cyp1a1 in vivo is reflective of AhR binding and activation, 147 compounds were evaluated for their ability to transform the AhR into a DNA-binding complex in vitro, induce expression of a DRE-driven reporter gene in rat H4L1.1c4 cells, and bind to the rat AhR in vitro. Of the 147 compounds that were evaluated in vitro, only 9 compounds showed significant activity in all 3 in vitro assays and significantly induced Cyp1a1 in vivo (Table 1). This includes the known AhR ligands 3-MC, BNF, and 
-naphthoflavone, which have been shown previously to be active in these assays. The other six compounds are approved for use by the FDA for a variety of indications, including omeprazole (Prilosec), nimodipine (Nimotop), leflunomide (Arava), flutamide (Eulexin), mexiletine (Mexitil), and atorvastatin (Lipitor) (Fig. 4). The most potent AhR agonist identified was leflunomide, a pyrimidine synthesis inhibitor indicated for rheumatoid arthritis, which induced luciferase activity as great as 1 nM TCDD, and completely displaced [3H]TCDD from the AhR (Table 1). Nimodipine, a calcium-channel blocker indicated for subarachnoid hemorrhage, and flutamide, an androgen receptor antagonist indicated for prostate cancer, also competitively displaced more than 90% of [3H]TCDD from the AhR. Omeprazole, previously believed to not bind the rat or human receptor (Daujat et al., 1992; Backlund et al., 1997) was found to displace approximately 50% of TCDD from the rat AhR and induced AhR transformation as determined by EMSA. Atorvastatin and mexiletine had weaker effects on luciferase activity (<20% of TCDD) and displaced less than 33% of TCDD from the AhR (Table 1). In contrast, indomethacin was weakly positive in all three in vitro assays yet did not significantly induce Cyp1a1 in vivo, nor did it consistently induce Cyp1a2, Ugt1a1, or Nqo1 (Supplementary Table S1). These results indicate that in vivo Cyp1a1 induction is a sensitive (9 of 10) indicator of AhR agonist activity, which is consistent with current understanding of AhR-mediated Cyp1a1 regulation (Fig. 5).
|
|
The agonist effects of leflunomide, nimodipine, and flutamide were further tested using the reporter gene assay, in which H4L1.1c4 cells were treated with increasing concentrations of compounds up to 10 µM (Fig. 6A). Leflunomide was the most potent among the three compounds and induced luciferase activity to a significantly greater level than that of TCDD. Based on the dose-response data, leflunomide had an EC50 value of 0.17 µM, which was approximately 2700-fold higher than that of TCDD (EC50 = 6.2 x 10–5 µM). Flutamide and nimodipine had EC50 values of 0.46 and 0.77 µM, respectively. Full dose-response curves could not be generated for omeprazole, mexiletine, and atorvastatin because of their relatively weak luciferase inducing potency.
|
Specificity of in Vivo Cyp1a1 Induction for Identifying AhR Agonists. Of the 137 parent compounds that were not consistently active in all 3 in vitro assays, 81 were found to significantly induce Cyp1a1 in vivo, thus indicating a high rate of false positives (59%) (Fig. 5). Of the 81 false positives, a number of compounds significantly induced Cyp1a2, Ugt1a1, and Nqo1 gene expression concurrently with Cyp1a1, thus suggesting activation through the classic AhR signaling pathway. These compounds included albendazole (days 1 and 3), rabeprozole (days 1, 3, and 5), safrole (days 1 and 3), melatonin (days 1 and 3), phenothiazine (days 1, 3, and 5), and sulindac (day 1) (Fig. 7A). Although metabolic activation may be necessary for in vivo AhR agonist activity for these compounds, there were a number of compounds that induced Cyp1a1 more than 10-fold but did not significantly induce Cyp1a2, Ugt1a1, or Nqo1; induce significant luciferase activity; or transform the AhR into a DNA binding form. These compounds were not tested in the AhR binding assay and included lovastatin, 1-naphthylisothiocyanate, eperisone, carvedilol, and zileuton (Fig. 7B). Other compounds that significantly induced luciferase activity and Cyp1a1 more than 10-fold but failed to stimulate the transformation of the AhR into a DNA binding form were also not tested in the binding assay. Notable compounds in this group include the corticosteroids dexamethasone and fludrocortisone in liver, prednisolone in heart, benoxaprofen and fenoprofen in liver, and cadmium chloride in kidney (Fig. 7C).
|
|
Discussion |
|---|
|
TopAbstractMaterials and MethodsResultsDiscussionReferences |
|---|
). AhR agonist activity for the major metabolite of safrole, 4-allyl-1,2-dihydroxybenzene, or others has not been reported, although Cyp1a1 induction has been observed previously for safrole, isosafrole, and related metabolites in mice (Cook and Hodgson, 1985; Lewandowski et al., 1990). Although melatonin is inactive as an AhR agonist in vitro (Fig. 7A) (Heath-Pagliuso et al., 1998), potential active metabolites of melatonin include 6-hydroxymelatonin, which is produced in humans by CYP1A1, CYP1A2, and CYP1B1 (Ma et al., 2005), thereby suggesting an autoinduction mechanism.
Numerous compounds have been reported to induce Cyp1a1 that do not seem to compete with TCDD for binding to the AhR, including thiazolium compounds, retinoids, carotenoids, benzimidazoles, carbamates, and aminoquinoline (Daujat et al., 1992; Aix et al., 1994; Lesca et al., 1995; Gradelet et al., 1997; Ledirac et al., 1997; Fontaine et al., 1999). Although the lack of in vitro AhR binding for the many Cyp1a1 inducers may result from technical limitations of the binding assays (Denison et al., 1998; Denison and Nagy, 2003), it has also been suggested that many of these compounds may induce Cyp1a1 through multiple modes of indirect AhR activation. For instance, a compound treatment may cause induction of endogenous metabolites or signaling molecules that regulate AhR. Aspartate aminotransferase has been shown to convert the proagonist L-tryptophan into a variety of AhR agonists (Bittinger et al., 2003). Furthermore, Cyp1a1 is inducible in the absence of exogenous ligand under conditions of hyperoxia (Okamoto et al., 1993), shear stress (Mufti and Shuler, 1996), and undefined serum factors (Guigal et al., 2000). Compound-induced production of endogenous ligands such as tryptophan metabolites or prostaglandins and other bioactive lipids that have been identified as AhR agonists (Schaldach et al., 1999; Seidel et al., 2001) may also be involved, although these hypotheses have yet to be confirmed. A more thorough understanding of these possible endogenous ligands and their levels in response to compound treatment may shed some light on this possibility.
There are data that support a role for numerous protein tyrosine kinases and mitogen-activated protein kinases in modulating AhR activity (Chen and Tukey, 1996; Backlund et al., 1997); however, the evidence thus far suggests that these kinases facilitate and/or amplify the functionality of the AhR rather than modulate Cyp1a1 independent of the AhR. The cooperative effects of phosphorylation and ligand binding to the AhR may result in in vivo expression of AhR-regulated genes being more sensitive than reporter gene-based or cell-free assays for detecting weak or transient ligands. This is supported by evidence showing differential sensitivity of Cyp1a1 induction to tyrosine kinase inhibitors in response to the weak ligand omeprazole relative to a high-affinity ligand like 3-MC (Lemaire et al., 2004). Differences in the inducibility of the native Cyp1a1 promoter in vivo and the DRE-regulated reporter construct in vitro may exist, although we know of no examples of bona fide agonists that fail to activate the DRE-regulated construct. Assay conditions may also make it difficult for in vitro assays to detect the ability of weak-affinity ligands to displace TCDD from the receptor given the strong affinity of TCDD (Kd values in the picomolar range). Although other studies have reported that omeprazole is unable to displace TCDD from the receptor (Daujat et al., 1992; Backlund et al., 1997), we detected significant activity in all three in vitro assays for omeprazole (Table 1), suggesting that the conditions used in our assays are more sensitive than those used by others. To this end, reports of AhR-independent induction of Cyp1a1 by chemicals have subsequently been reconsidered through the use of a more sensitive binding assay (Denison et al., 1998; Denison and Nagy, 2003).
Under certain treatment conditions, the expression of Cyp1a1 was induced, whereas other DRE-regulated genes were not. Compounds with this profile deviate from the classic mechanism of AhR binding and transcriptional activation via DREs. Most notable among these compounds are the corticosteroids (Fig. 7C). Dexamethasone has been shown previously to induce Cyp1a1 at high concentrations and potentiate TCDD-induced expression in a glucocorticoid receptor and protein synthesis-dependent manner (Lai et al., 2004). Up-regulation or activation of certain transcription factors such as retinoid X receptor, PGC-1, and hepatic nuclear factor 4-, or calcium-dependent calpain may also contribute indirectly to Cyp1a1 induction (Gradelet et al., 1997; Dale and Eltom, 2006; Martinez-Jimenez et al., 2006). PGC-1 was observed to be induced by most heart and kidney treatments concurrent with Cyp1a1 up-regulation (data not shown) in which Cyp1a2, NQO1, and Ugt1a1 were not induced (Fig. 2). Two peroxisome proliferator-activated receptor (PPAR) response elements were found to mediate induction of human Cyp1a1 in response to PPAR- agonists (Seree et al., 2006). PGC-1 positively regulates PPAR- activity, thus suggesting that these transcription factors synergize to induce rat Cyp1a1 in a similar manner (Fig. 2B). It is interesting that treatment of ischemic rats with a PPAR- ligand has been shown to be cardioprotective as a result of nitric oxide production (Bulhak et al., 2006), which has also been shown to repress Cyp1a2 mRNA (Mulero-Navarro et al., 2003). Although definitive proof is still lacking, these findings and the observations in Fig. 2 suggest a model whereby rat Cyp1a1 is specifically induced by PGC-1/PPAR- in the heart with concomitant production of nitric oxide and a resulting down-regulation of Cyp1a2.
The lack of specificity of Cyp1a1 as a biomarker of AhR activation raises significant concern over the use of Cyp1a1 and its related enzyme activities (i.e., ethoxyresorufin-O-deethylase activity) to evaluate the potential of compounds or mixtures to activate the AhR. Given the lack of specificity, an overestimation of AhR activation potential and calculated toxicity equivalents may result from the strict reliance on Cyp1a1 mRNA, protein, or enzyme activity alone without the use of more specific assays or a combination of functional or binding assays to confirm the dependence on AhR binding and transcriptional activation. With respect to estimates of dioxin-like toxicity, a rich body of literature indicates that metabolically persistent halogenated ligands of the AhR cause sustained activation of the receptor and result in a wide spectrum of toxic responses similar to TCDD, whereas metabolically labile, nonhalogenated AhR ligands do not typically produce dioxin-like toxicities in animal studies. Recent studies in fish have demonstrated that inhibition of Cyp1a1-dependent metabolism of these labile AhR agonists can result in dioxin-like toxicity because of the increased persistence of the chemical (Wassenberg and Di Giulio, 2004). These results suggest that whereas binding and activation of the AhR are necessary prerequisite events for AhR-dependent dioxin-like toxicity, the actual occurrence of toxicity requires both continual presence of the AhR agonist and persistent activation of the AhR signaling pathway. In the current study, through a combination of in vivo and in vitro assays, a number of weak AhR ligands were identified, including nimodipine, leflunomide, flutamide, omeprazole, mexiletine, and atorvastatin. These compounds, which are approved for use by the U.S. FDA, do not produce dioxin-like toxicities in rats, and there is no evidence for chloracne, immunosuppression, or other adverse dioxin-like effects in exposed humans. This could be due to both their reduced potency relative to TCDD and/or their rapid rate of clearance from the body relative to persistent halogenated ligands. It would seem that the toxicological consequences of transient or weak receptor activation are qualitatively and quantitatively distinct from persistent activation by metabolically stable and potent ligands.
Several lines of evidence presented in the current study are consistent with the conclusion that the induction of rat Cyp1a1 is a sensitive but not specific indicator of AhR binding and activation. Furthermore, the induction of Cyp1a1 and activation of AhR is not synonymous with dioxin-like toxicity in the rat for nonhalogenated or metabolically labile compounds. Second-tier hazard-identification strategies such as the in vitro tests used herein should be considered for assessing exposure and toxicity related to AhR activation.
|
Footnotes |
|---|
ABBREVIATIONS: AhR, aryl hydrocarbon receptor; DRE, dioxin-response element; HAH, halogenated aromatic hydrocarbon; TCDD, 2,3,7,8-tetrachlorodibenzo-p-dioxin; Cyp1a1, cytochrome P4501a1; Cyp1a2, cytochrome P4501a2; Ugt1a1, UDP-glucuronosyltransferase 1a1; Nqo1, NAD(P)H:quinone oxidoreductase 1; 3-MC, 3-methylcholanthrene; BNF, -naphthoflavone; EMSA, electrophoretic mobility shift assay; HEDG buffer, HEPES/EDTA/dithiothreitol/glycerol; DMSO, dimethyl sulfoxide; FDA, Food and Drug Administration; PGC-1, peroxisome proliferator activated receptor- coactivator; PPAR, peroxisome proliferator-activated receptor; 5-HT2B, 5-hydroxytryptamine-2B; BW-723C86, 1-[5-(2-thienylmethoxy)-1H-3-indolyl]propan-2-amine HCl.
The online version of this article (available at http://molpharm.aspetjournals.org) contains supplemental material. 
Address correspondence to: Dr. Mark R. Fielden, Iconix Biosciences, Inc., 325 E. Middlefield Road, Mountain View, CA 94043. E-mail: mfielden{at}iconixbiosciences.com
|
References |
|---|
|
TopAbstractMaterials and MethodsResultsDiscussionReferences |
|---|
Backlund M, Johansson I, Mkrtchian S, and Ingelman-Sundberg M (1997) Signal transduction-mediated activation of the aryl hydrocarbon receptor in rat hepatoma H4IIE cells. J Biol Chem 272: 31755–31763.
Backlund M, Weidolf L, and Ingelman-Sundberg M (1999) Structural and mechanistic aspects of transcriptional induction of cytochrome P450 1a1 by benzimidazole derivatives in rat hepatoma H4IIE cells. Eur J Biochem 261: 66–71.[Medline]
Behnisch PA, Hosoe K, Brouwer A, and Sakai S (2002) Screening of dioxin-like toxicity equivalents for various matrices with wildtype and recombinant rat hepatoma H4IIE cells. Toxicol Sci 69: 125–130.
Behnisch PA, Hosoe K, and Sakai S (2001) Bioanalytical screening methods for dioxins and dioxin-like compounds a review of bioassay/biomarker technology. Environ Int 27: 413–439.[CrossRef][Medline]
Bittinger MA, Nguyen LP, and Bradfield CA (2003) Aspartate aminotransferase generates proagonists of the aryl hydrocarbon receptor. Mol Pharmacol 64: 550–556.
Bulhak AA, Sjoquist PO, Xu CB, Edvinsson L, and Pernow J (2006) Protection against myocardial ischaemia/reperfusion injury by PPAR-alpha activation is related to production of nitric oxide and endothelin-1. Basic Res Cardiol 101: 244–252.[CrossRef][Medline]
Chen YH and Tukey RH (1996) Protein kinase C modulates regulation of the Cyp1a1 gene by aryl hydrocarbon receptor. J Biol Chem 271: 26261–26266.
Cook JC, and Hodgson E (1985) The induction of cytochrome P450 by isosafrole and related methylenedioxyphenyl compounds. Chem Biol Interact 54: 299–315.[CrossRef][Medline]
Dale Y and Eltom SE (2006) The induction of Cyp1a1 by oltipraz is mediated through calcium-dependent-calpain. Toxicol Lett 166: 150–159.[CrossRef][Medline]
Daujat M, Peryt B, Lesca P, Fourtanier G, Domergue J, and Maurel P (1992) Omeprazole, an inducer of human Cyp1a1 and 1a2, is not a ligand for the Ah receptor. Biochem Biophys Res Commun 188: 820–825.[CrossRef][Medline]
Delescluse C, Lemaire G, de Sousa G, and Rahmani R (2000) Is Cyp1a1 induction always related to AHR signaling pathway? Toxicology 153: 73–82.[CrossRef][Medline]
Denison MS and Nagy SR (2003) Activation of the aryl hydrocarbon receptor by structurally diverse exogenous and endogenous chemicals. Ann Rev Pharmacol Toxicol 43: 309–334.[CrossRef][Medline]
Denison MS, Pandini A, Nagy SR, Baldwin EP, and Bonati L (2002) Ligand binding and activation of the Ah receptor. Chem Biol Interact 141: 3–24.[CrossRef][Medline]
Denison MS, Phelan D, Winter MG, and Ziccardi MH (1998) Carbaryl, a carbamate insecticide, is a ligand for the hepatic Ah (dioxin) receptor. Toxicol Appl Pharmacol 152: 406–414.[CrossRef][Medline]
Fontaine F, Delescluse C, de Sousa G, Lesca P, and Rahmani R (1999) Cytochrome 1a1 induction by primaquine in human hepatocytes and HepG2 cell: absence of binding to the aryl hydrocarbon receptor. Biochem Pharmacol 57: 255–262.[CrossRef][Medline]
Ganter B, Tugendreich S, Pearson CI, Ayanoglu E, Baumhueter S, Bostian KA, Brady L, Browne LJ, Calvin JT, Day GJ, et al. (2005) Development of a large-scale chemogenomics database to improve drug candidate selection and to understand mechanisms of chemical toxicity and action. J Biotechnol 119: 219–244.[CrossRef][Medline]
Garrison PM, Tullis K, Aarts JM, Brouwer A, Giesy JP, and Denison MS (1996) Species-specific recombinant cell lines as bioassay systems for the detection of 2,3,7,8-tetrachlorodibenzo-p-dioxin-like chemicals. Fundam Appl Toxicol 30: 194–203.[CrossRef][Medline]
Gonzalez FJ, Fernandez-Salguero P, and Ward JM (1996) The role of the aryl hydrocarbon receptor in animal development, physiological homeostasis and toxicity of TCDD. J Toxicol Sci 21: 273–277.[Medline]
Gradelet S, Astorg P, Pineau T, Canivenc MC, Siess MH, Leclerc J, and Lesca P (1997) Ah receptor-dependent Cyp1a induction by two carotenoids, canthaxanthin and carotenal, with no affinity for the TCDD binding site. Biochem Pharmacol 54: 307–315.[CrossRef][Medline]
Guigal N, Seree E, Bourgarel-Rey V, and Barra Y (2000) Induction of Cyp1a1 by serum independent of AhR pathway. Biochem Biophys Res Commun 267: 572–576.[CrossRef][Medline]
Heath-Pagliuso S, Rogers WJ, Tullis K, Seidel SD, Cenijn PH, Brouwer A, and Denison MS (1998) Activation of the Ah receptor by tryptophan and tryptophan metabolites. Biochemistry 37: 11508–11515.[CrossRef][Medline]
Lai KP, Wong MH, and Wong CK (2004) Modulation of AhR-mediated CYP1A1 mRNA and EROD activities by 17beta-estradiol and dexamethasone in TCDD-induced H411E cells. Toxicol Sci 78: 41–49.
Ledirac N, Delescluse C, de Sousa G, Pralavorio M, Lesca P, Amichot M, Berge JB, and Rahmani R (1997) Carbaryl induces CYP1A1 gene expression in HepG2 and HaCaT cells but is not a ligand of the human hepatic Ah receptor. Toxicol Appl Pharmacol 144: 177–182.[CrossRef][Medline]
Lemaire G, Delescluse C, Pralavorio M, Ledirac N, Lesca P, and Rahmani R (2004) The role of protein tyrosine kinases in Cyp1a1 induction by omeprazole and thiabendazole in rat hepatocytes. Life Sci 74: 2265–2278.[CrossRef][Medline]
Lesca P, Peryt B, Larrieu G, Alvinerie M, Galtier P, Daujat M, Maurel P, and Hoogenboom L (1995) Evidence for the ligand-independent activation of the Ah receptor. Biochem Biophys Res Commun 209: 474–482.[CrossRef][Medline]
Lewandowski M, Chui YC, Levi PE, and Hodgson E (1990) Differences in induction of hepatic cytochrome P450 isozymes by mice in eight methylenedioxyphenyl compounds. J Biochem Toxicol 5: 47–55.[CrossRef][Medline]
Ma XC, Idle JR, Krausz KW, and Gonzalez FJ (2005) Metabolism of melatonin by human cytochrome P450. Drug Metab Dispos 33: 489–494.
Martinez-Jimenez CP, Castell JV, Gomez-Lechon MJ, and Jover R (2006) Transcriptional activation of Cyp2c9, Cyp1a1 and Cyp1a2 by hepatocyte nuclear factor 4 requires coactivators peroxisomal proliferator activated receptor- coactivator 1 and steroid receptor coactivator 1. Mol Pharmacol 70: 1681–1692.
Mufti NA and Shuler ML (1996) Possible role of arachidonic acid in stress-induced cytochrome P4501A1 activity. Biotechnol Prog 12: 847–854.[CrossRef][Medline]
Mulero-Navarro S, Santiago-Josefat B, Pozo-Guisado E, Merino JM, and Fernandez-Salguero PM (2003) Down-regulation of CYP1A2 induction during the maturation of mouse cerebellar granule cells in culture: role of nitric oxide accumulation. Eur J Neurosci 18: 2265–2272.[CrossRef][Medline]
Okamoto T, Misuhashi M, Fujuta I, Shindhu RK, and Kikkawa Y (1993) Induction of cytochrome P450 1A1 and 1A2 by hyperoxia. Biochem Biophys Res Commun 197: 878–885.[CrossRef][Medline]
Phelan D, Winter GM, Rogers WJ, Lam JC, and Denison MS (1998) Activation of the Ah receptor signal transduction pathway by bilirubin and biliverdin. Arch Biochem Biophys 357: 155–163.[CrossRef][Medline]
Schaldach CM, Riby J, and Bjeldanes LF (1999) Lipoxin A4: a new class of ligand for the Ah receptor. Biochemistry 38: 7594–7600.[CrossRef][Medline]
Seidel SD, Winters GM, Rogers WJ, Ziccardi MH, Li V, Keser B, and Denison MS (2001) Activation of the Ah receptor signaling pathway by prostaglandins. J Biochem Mol Toxicol 15: 187–196.[CrossRef][Medline]
Seree E, Villard PH, Pascussi JM, Pineau T, Maurel P, Nguyen QB, Fallone F, Martin PM, Champion S, Lacarelle B, et al. (2006) Evidence for a new human CYP1A1 regulation pathway involving PPAR-alpha and 2 PPRE sites. Gastroenterology 127: 1436–1445.[CrossRef]
Shih H, Pickwell GV, Guenette DK, Bilir B, and Ouattrochi LC (1999) Species differences in hepatocyte induction of cyp1a1 and cyp1a2 by omeprazole. Hum Exp Toxicol 18: 95–105.
Wassenberg DM and Di Giulio RT (2004) Synergistic embryotoxicity of polycyclic aromatic hydrocarbon aryl hydrocarbon receptor agonists with cytochrome P4501A inhibitors in Fundulus heteroclitus. Environ Health Perspect 112: 1658–1664.[Medline]
This article has been cited by other articles:
|
|
 |
|
  B. P. Lawrence, M. S. Denison, H. Novak, B. A. Vorderstrasse, N. Harrer, W. Neruda, C. Reichel, and M. Woisetschlager Activation of the aryl hydrocarbon receptor is essential for mediating the anti-inflammatory effects of a novel low-molecular-weight compound Blood, August 15, 2008; 112(4): 1158 - 1165. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
 |
 |
 |
|
 |
 |
 |
