The alkaloid rutaecarpine is a selective inhibitor of cytochrome P450 1A in mouse and human liver microsomes.

Rutaecarpine, evodiamine, and dehydroevodiamine are quinazolinocarboline alkaloids isolated from a traditional Chinese medicine, Evodia rutaecarpa. The in vitro effects of these alkaloids on cytochrome P450 (P450)-catalyzed oxidations were studied using mouse and human liver microsomes. Among these alkaloids, rutaecarpine showed the most potent and selective inhibitory effect on CYP1A-catalyzed 7-methoxyresorufin O-demethylation (MROD) and 7-ethoxyresorufin O-deethylation (EROD) activities in untreated mouse liver microsomes. The IC(50) ratio of EROD to MROD was 6. For MROD activity, rutaecarpine was a noncompetitive inhibitor with a K(i) value of 39 +/- 2 nM. In contrast, rutaecarpine had no effects on benzo[a]pyrene hydroxylation (AHH), aniline hydroxylation, and nifedipine oxidation (NFO) activities. In human liver microsomes, 1 microM rutaecarpine caused 98, 91, and 77% decreases of EROD, MROD, and phenacetin O-deethylation activities, respectively. In contrast, less than 15% inhibition of AHH, tolbutamide hydroxylation, chlorzoxazone hydroxylation, and NFO activities were observed in the presence of 1 microM rutaecarpine. To understand the selectivity of inhibition of CYP1A1 and CYP1A2, inhibitory effects of rutaecarpine were studied using liver microsomes of 3-methylcholanthrene (3-MC)-treated mice and Escherichia coli membrane expressing bicistronic human CYP1A1 and CYP1A2. Similar to the CYP1A2 inhibitor furafylline, rutaecarpine preferentially inhibited MROD more than EROD and had no effect on AHH in 3-MC-treated mouse liver microsomes. For bicistronic human P450s, the IC(50) value of rutaecarpine for EROD activity of CYP1A1 was 15 times higher than the value of CYP1A2. These results indicated that rutaecarpine was a potent inhibitor of CYP1A2 in both mouse and human liver microsomes.     

Benzyl butyl phthalate induces necrosis by AhR mediation of CYP1B1 expression in human granulosa cells

We investigated the signaling pathway of the arylhydrocarbon receptor (AhR) on HO23 cells (immortalized human granulosa cells (hGC)) mediated by benzyl butyl-phthalate (BBP). BBP (1 μM) significantly increased the mRNA and protein levels of AhR, aryl hydrocarbon receptor nuclear translocator (ARNT) and cytochrome-P450 (CYP)1B1 in HO23 cells. Treatment with 3',4'-dimethoxyflavone (3',4'-DMF) or AhR siRNA significantly reduced AhR and CYP1B1, but CYP1A1 was not affected by 3',4'-DMF or AhR siRNA, suggesting that increases in CYP1A1 may not regulated by AhR. BBP induced the AhR fusion protein to localize and accumulate around the nucleus, and AhR heterodimerization with ARNT was observed in the nucleus by immunoprecipitation. Chromatin immunoprecipitation and reporter assays revealed the effect of BBP on CYP1B1, but not CYP1A1. Necrosis was significantly increased in HO23 cells after BBP treatment, and 3',4'-DMF, AhR siRNA or CYP1B1 siRNA knockdown blocked this phenomenon. These data suggest that BBP-induced HO23 cell necrosis is AhR and CYP1B1 dependent.     

Comparative study of aryl hydrocarbon receptor ligand activities of six chemicals in vitro and in vivo

The aryl hydrocarbon receptor (AhR) ligand activities of six known AhR ligands were compared in vivo and in vitro. The in vivo ligand activity was estimated in terms of induction of cytochrome P450 1A1/2 activities, i.e., ethoxyresorufin-O-dealkylase (EROD) and methoxyresorufin-O-dealkylase (MROD) activities, and in vitro ligand activity was evaluated with a recombinant yeast reporter gene assay. The test chemicals were 3-methylcholanthrene (MC), β-naphthoflavone (β-NF), indirubin, indigo, 3,3′-diindolylmethane (DIM) and diphenyl-p-phenylenediamine (DPPD). The first four showed potent AhR ligand activity in vitro, comparable with that of 2,3,7,8-tetrachlorodibenzo-p-dioxin, while DIM and DPPD showed weaker activity. Administration of MC and β-NF to mice caused significant induction of EROD and MROD activities, while indirubin, indigo and DIM also induced these activities, but less potently. DPPD also induced the activities, but was toxic at higher doses. These enhancing effects were lost or greatly reduced in Ahr-null mice (Ahr ^−/−). Our results suggest that EROD and MROD activity assays are useful for evaluating the AhR ligand activity of chemicals in vivo, where the biodynamics of the chemicals plays an important role.     

Mechanisms of action of two different natural mixtures of persistent organic pollutants (POPs) in ovarian follicles

1. The present work investigated the effects of two different natural mixtures on aryl hydrocarbon receptor (AhR) and oestrogen receptor (ER)β protein levels, as well as on the activity of cytochrome P450 (CYP) 1A1 and CYP2B. Consequently, the authors observed the effects of these mixtures on gonadotropine-stimulated steroid secretion by ovarian follicles. The natural mixtures that were studied were ‘Mjosa’ extracted from burbot liver, which contains a high level of PBDEs, and ‘Marine mix’, extracted from Atlantic cod liver, which contains a high level of polychlorinated biphenyls (PCBs).

2. Follicular cells were exposed in vitro to ‘Marine mix’ and ‘Mjosa mix’ at doses of 3.6 and 1.4 μg ml^?1, respectively. Media were collected and used for steroid analysis and cell viability assays. Cells were used to estimate aromatase activity (CYP19), AhR and ER protein levels, and CYP1A1 and CYP2B1 activity.

3. Western blot analysis indicated down-regulation of AhR by ‘Marine mix’ and down-regulation of ERβ by Mjosa mix. Up-regulation of CYP1A1 expression and activity were seen following treatment with Marine mix, but not Mjosa mix. Increased CYP2B1 activity was noted after treatment with both ‘Marine mix’ and Mjosa mix. Both mixtures increased luteinizing hormone (LH)-stimulated progesterone and testosterone secretion, follicle-stimulating hormone (FSH)-stimulated oestradiol secretion, and CYP19 activity.

4. These results suggest that: (1) ‘Marine mix’ is a mixed-type CYP inducer; (2) ‘Mjosa mix’ is an inducer of ERβ and CYP2B; and (3) both ‘Marine mix’ and ‘Mjosa mix’ stimulate aromatase activity as a consequence of oestradiol secretion through activation of CYP19

     

Regulation of CYP1A1 gene expression by the antioxidant tert-butylhydroquinone.

CYP1A1, a major phase I enzyme, plays an important role in the metabolism of polycyclic aromatic hydrocarbons and in the chemical activation of xenobiotics to carcinogenic derivatives. The phenolic antioxidant tert-butylhydroquinone (tBHQ), often used as a food preservative, is generally considered to act only as a mono-functional inducer of phase II enzymes, thereby exerting chemo-protection. However, we recently observed that tBHQ elevated the activity of an aryl hydrocarbon receptor (AhR) response element (DRE)-driven luciferase reporter in human colon carcinoma cells (Caco-2). Therefore, we studied the effects of tBHQ on the activity of a DRE-driven reporter, CYP1A1 mRNA expression, and CYP1A enzyme activity in Caco-2 cells and human HepG2 hepatoma cells. We found tBHQ caused induction of reporter activity and CYP1A1 expression and activity in Caco-2 and HepG2 cells. Moreover, tBHQ combined with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) increased reporter activity and mRNA expression in Caco-2 cells in an additive manner. By contrast, tBHQ decreased TCDD-mediated induction of reporter activity and CYP1A1 mRNA expression in HepG2 cells. Resveratrol, an AhR antagonist, repressed the induction of CYP1A1 by tBHQ. Cotransfection of HepG2 cells with a dominant negative AhR nuclear translocator mutant abolished the tBHQ-induced CYP1A1 reporter activity. These findings indicate that CYP1A1 may be induced by the antioxidant tBHQ via an AhR-dependent mechanism.     

Dioxin-mediated up-regulation of aryl hydrocarbon receptor target genes is dependent on the calcium/calmodulin/CaMKIalpha pathway

Regulation of genes targeted by the ligand-activated aryl hydrocarbon receptor (AhR) has been shown to be controlled by calcium (Ca(2+)) changes induced by AhR agonists such as the environmental contaminant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). The present study was designed to characterize this link between Ca(2+) and the AhR pathway. We report that fast elevation of intracellular Ca(2+) in TCDD-exposed mammary MCF-7 cells was associated with transient enhanced activity of the Ca(2+)/calmodulin (CaM)-dependent protein kinase (CaMK) pathway. Chemical inhibition of this pathway using the CaM antagonist W7 or the CaMK inhibitor KN-93 strongly reduced TCDD-mediated induction of the AhR target gene CYP1A1. Small interfering RNA (siRNA)-mediated knockdown expression of CaMKIalpha, one of the CaMK isoforms, similarly prevented CYP1A1 up-regulation. Both KN-93 and siRNA targeting CaMKIalpha were found to abolish TCDD-mediated activation of CYP1A1 promoter and TCDD-triggered nuclear import of AhR, a crucial step of the AhR signaling pathway. TCDD-mediated inductions of various AhR targets, such as the drug metabolizing CYP1B1, the cytokine interleukin-1beta, the chemokines interleukin-8 and CCL1, the adhesion molecule beta7 integrin, and the AhR repressor, were also prevented by KN-93 in human macrophages. Taken together, these data identified the Ca(2+)/CaM/CaMKIalpha pathway as an important contributing factor to AhR-mediated genomic response.     

Polycyclic aromatic hydrocarbons activate CYP3A4 gene transcription through human pregnane X receptor

Aryl hydrocarbon receptor (AhR) activators have been shown to induce members of the cytochrome P450 (P450) 1 family. Here we demonstrate that the AhR activators induce CYP3A4 through human pregnane X receptor (PXR). AhR activators, polycyclic aromatic hydrocarbons (PAHs) and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) increased CYP3A4 reporter activity and CYP3A4 mRNA expression in HepG2 cells. The CYP3A4 reporter activity was also increased by treatment with cigarette tar. The increased CYP3A4 reporter activity was clearly knocked down by the introduction of human PXR-small interfering RNA, but not by that of human AhR-small interfering RNA. The CYP3A4 reporter activity enhanced by overexpression of human PXR was further increased by treatment with PAHs and TCDD as well as by treatment with rifampicin. These results suggest that PAHs contained in cigarette smoke induce CYP3A4 in human liver.     

Transient induction of cytochromes P450 1A1 and 1B1 in MCF-7 human breast cancer cells by indirubin

The aryl hydrocarbon receptor (AhR), when activated by exogenous ligands such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), regulates expression of several phase I and phase II enzymes and is also involved in the regulation of cell proliferation. Several studies suggest that endogenous AhR ligand(s) may exist. One putative endogenous ligand is indirubin, which was recently identified in human urine and bovine serum. We determined the effect of indirubin in MCF-7 breast cancer cells on induction of the activities of cytochromes P450 (CYP) 1A1 and 1B1, as measured by estradiol and ethoxyresorufin metabolism, and on induction of the CYP1A1 and CYP1B1 mRNAs. With 4-hr exposure, the effects of indirubin and TCDD at 10nM on CYP activity were comparable, but the effects of indirubin, unlike those of TCDD, were transitory. Indirubin-induced ethoxyresorufin-O-deethylase activity was maximal by 6-9 hr post-exposure and had disappeared by 24 hr, whereas TCDD-induced activities remained elevated for at least 72 hr. The effects of indirubin on CYP mRNA induction were maximal at 3 hr. Indirubin was metabolized by microsomes containing cDNA-expressed human CYP1A1 or CYP1B1. The potency of indirubin was comparable to that of TCDD in a CYP1B1-promoter-driven luciferase assay, when MCF-7 cells were co-exposed to the AhR ligands together with the CYP inhibitor, ellipticine. Thus, if indirubin is an endogenous AhR ligand, then AhR-mediated signaling by indirubin is likely to be transient and tightly controlled by the ability of indirubin to induce CYP1A1 and CYP1B1, and hence its own metabolism.     

p-Anilinoaniline enhancement of dioxin-induced CYP1A1 transcription and aryl hydrocarbon receptor occupancy of CYP1A1 promoter: role of the cell cycle

The aryl hydrocarbon receptor (AhR) is targeted by ubiquitination for degradation by the proteasome shortly after its activation by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). In silico screening identified p-anilinoaniline (pAA) as a putative inhibitor of an E2 ligase that partners with an E3 ligase implicated in AhR ubiquitination. We investigated whether pAA could modify AhR-dependent activation of its target gene CYP1A1. pAA (1-200 μM) alone did not affect AhR content, or stimulate CYP1A1 mRNA accumulation in human mammary epithelial MCF10A cultures. However, pretreatment with ≥100 μM pAA suppressed TCDD-induced CYP1A1 activation and AhR degradation via its functioning as an AhR antagonist. At a lower concentration (25 μM), pAA cotreatment increased TCDD-induced CYP1A1 mRNA accumulation, without inhibiting AhR turnover or altering CYP1A1 mRNA half-life. Whereas TCDD alone did not affect MCF10A proliferation, 25 μM pAA was cytostatic and induced a G(1) arrest that lasted ～7 h and induced an S phase arrest that peaked 5 to 8 h later. TCDD neither affected MCF10A cell cycle progression nor did it alter pAA effects on the cell cycle. The magnitude of CYP1A1 activation depended upon the time elapsed between pAA pretreatment and TCDD addition. Maximal AhR occupancy of the CYP1A1 promoter and accumulation of CYP1A1 heterogeneous nuclear RNA and mRNA occurred when pAA-pretreated cultures were exposed to TCDD in late G(1) and early/mid S phase. TCDD-mediated induction of CYP2S1 was also cell cycle-dependent in MCF10A cultures. Similar studies with HepG2 cultures indicated that the cell cycle dependence of CYP1A1 induction is cell context-dependent.     

JNK inhibitor SP600125 is a partial agonist of human aryl hydrocarbon receptor and induces CYP1A1 and CYP1A2 genes in primary human hepatocytes

SP600125, a specific inhibitor of c-Jun-N-Terminal kinase (JNK), was reported as a ligand and antagonist of aryl hydrocarbon receptor (AhR) [Joiakim A, Mathieu PA, Palermo C, Gasiewicz TA, Reiners Jr JJ. The Jun N terminal kinase inhibitor SP600125 is a ligand and antagonist of the aryl hydrocarbon receptor. Drug Metab Dispos 2003;31(11):1279-82]. Here we show that SP600125 is not an antagonist but a partial agonist of human AhR. SP600125 significantly induced CYP1A1 and CYP1A2 mRNAs in primary human hepatocytes and CYP1A1 mRNA in human hepatoma cells HepG2. This effect was abolished by resveratrol, an antagonist of AhR. Consistent with the recent report, SP600125 dose-dependently inhibited CYP1A1 and CYP1A2 genes induction by a prototype AhR ligand 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in human hepatocytes. Moreover, SP600125 displayed typical behavior of a partial agonist in HepG2 cells transiently transfected with a reporter plasmid containing two inverted repeats of the dioxin responsive element or with a plasmid containing 5'-flanking region of human CYP1A1 gene. SP600125 transactivated the reporter plasmids with EC(50) of 0.005 and 1.89 microM, respectively. On the other hand, TCDD-dependent transactivation of the reporter plasmids was inhibited by SP600125 with IC(50) values of 1.54 and 2.63 microM, respectively. We also tested, whether the effects of SP600125 are due to metabolism. Using liquid chromatography/mass spectrometry approach, we observed formation of two minor monohydroxylated metabolites of SP600125 in human hepatocytes, human liver microsomes but not in HepG2 cells. These data imply that biotransformation is not responsible for the effects of SP600125 on AhR signaling. In conclusion, we demonstrate that SP600125 is a partial agonist of human AhR, which induces CYP1A genes.     

Polycyclic aromatic hydrocarbons stimulate human CYP3A4 promoter activity via PXR

Metabolic activation of polycyclic aromatic hydrocarbons (PAH) is mediated mainly by cytochrome P₄₅₀ monooxygenases (CYP) CYP1A1, 1A2 and 1B1. Several PAH are known to induce these CYP via aryl hydrocarbon receptor (AhR) signaling. Recently, it was shown that the PAH benzo[a]pyrene (BaP) can induce CYP3A4 as well. The induction was suggested to be mediated by the pregnane X receptor (PXR) rather than AhR. Metabolism by CYP3A4 is only known for dihydrodiol metabolites of PAH but not for their parent compounds.     

Lack of CYP1A1 expression is involved in unresponsiveness of the human hepatoma cell line SK-HEP-1 to dioxin

The aryl hydrocarbon receptor (AhR) mediates a wide variety of toxic effects due to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). The human hepatoma cell line SK-HEP-1 expresses AhR and ARNT. However, TCDD failed to induce CYP1A1 and XRE-dependent reporter genes in these cells. Although CYP1A1 was not induced by TCDD exposure, both CYP1B1 and AhR repressor (AhRR) were constitutively expressed. The AhR antagonist alpha-naphthoflavone altered the basal level of XRE-dependent reporter gene expression dose-dependently. As our results suggested the activation of AhR signals by putative endogenous ligands, we established SK-HEP-1-derived cell lines that stably expressed CYP1A1. The inducibility of XRE-dependent reporter genes and CYP1B1 by TCDD was restored in these cells. Our findings demonstrated the presence of endogenous ligands in SK-HEP-1 cells due to the absence of the metabolizing enzyme CYP1A1, but not CYP1B1, which allowed the constitutive expression of AhR target genes.