Harmaline and harmalol inhibit the carcinogen-activating enzyme CYP1A1 via transcriptional and posttranslational mechanisms

Dioxins are known to cause several human cancers through activation of the aryl hydrocarbon receptor (AhR). Harmaline and harmalol are dihydro-β-carboline compounds present in several medicinal plants such as Peganum harmala. We have previously demonstrated the ability of P. harmala extract to inhibit TCDD-mediated induction of Cyp1a1 in murine hepatoma Hepa 1c1c7 cells. Therefore, the aim of this study is to examine the effect of harmaline and its main metabolite, harmalol, on dioxin-mediated induction of CYP1A1 in human hepatoma HepG2 cells. Our results showed that harmaline and harmalol at concentrations of (0.5-12.5μM) significantly inhibited the dioxin-induced CYP1A1 at mRNA, protein and activity levels in a concentration-dependent manner. The role of AhR was determined by the inhibition of the TCDD-mediated induction of AhR-dependent luciferase activity and the AhR/ARNT/XRE formation by both harmaline and harmalol. In addition, harmaline significantly displaced [(3)H]TCDD in the competitive ligand binding assay. At posttranslational level, both harmaline and harmalol decreased the protein stability of CYP1A1, suggesting that posttranslational modifications are involved. Moreover, the posttranslational modifications of harmaline and harmalol involve ubiquitin-proteasomal pathway and direct inhibitory effects of both compounds on CYP1A1 enzyme. These data suggest that harmaline and harmalol are promising agents for preventing dioxin-mediated effects.     

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.     

Transcriptional and posttranslational inhibition of dioxin-mediated induction of CYP1A1 by harmine and harmol

Dioxins are widespread environmental contaminants that induce the carcinogen-activating enzyme, cytochrome P450 1A1 (CYP1A1) through an aryl hydrocarbon receptor (AhR)-dependent mechanism. We previously demonstrated that harmine inhibits the dioxin-mediated induction of Cyp1a1 activity in murine hepatoma cells. Therefore, the aim of this study is to determine the effect of harmine and its main metabolite, harmol, on the dioxin-mediated induction of CYP1A1 in human HepG2 and murine Hepa 1c1c7 hepatoma cells. Our results showed that harmine and harmol significantly inhibited the dioxin-mediated induction of CYP1A1 at mRNA, protein, and activity levels in a concentration-dependent manner in human and murine hepatoma cells. Moreover, harmine and harmol inhibited the AhR-dependent luciferase activity and the activation and transformation of AhR using the electrophoretic mobility shift assay. In addition, harmine and harmol displaced [³H]TCDD in the competitive ligand binding assay. At posttranslational level, both harmine and harmol decreased the protein stability of CYP1A1, suggesting that posttranslational mechanism is involved. Furthermore, we demonstrated that the underlying mechanisms of the posttranslational modifications of both compounds involve ubiquitin-proteasomal pathway and direct inhibitory effects of CYP1A1 enzyme. We concluded that harmine and its metabolite, harmol, are new inhibitors of dioxin-mediated effects.     

Effect of mercury on aryl hydrocarbon receptor-regulated genes in the extrahepatic tissues of C57BL/6 mice

The individual toxic effects of aryl hydrocarbon receptors (AhR) ligands such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) or heavy metals typified by mercury (Hg²⁺) has been previously demonstrated. However, little is known about the combined toxic effects of TCDD and Hg²⁺in vivo. Therefore, we examined the effect of exposure to Hg²⁺ (2.5 mg/kg) in the absence and presence of TCDD (15 μg/kg) on the AhR-regulated genes using C57Bl/6 mice. Hg²⁺ alone did not affect kidney, lung, or heart Cyp1a1/1a2/1b1 mRNA levels. On the contrary, Hg²⁺ alone significantly induced kidney Cyp1a1/1a2/1b1 and lung Cyp1b1 protein and catalytic activities. Hg²⁺ also induced Nqo1, Gsta1, and HO-1 at the mRNA, protein, and activity levels in the kidney and heart but not in the lung. Upon co-exposure to Hg²⁺ and TCDD, Hg²⁺ significantly potentiated the TCDD-mediated induction of kidney and lung Cyp1a1/1a2/1b1 mRNA levels, while it decreased their kidney protein and catalytic activity and it increased their lung protein. In addition, Hg²⁺ potentiated the TCDD-mediated induction of Nqo1, Gsta1, and HO-1 at mRNA, protein and activity levels in all tissues. The present study demonstrates that Hg²⁺ modulates the constitutive and TCDD-induced AhR-regulated genes in a time-, tissue- and, AhR-regulated enzyme genes manner.     

The role of protein kinase C in regulation of TCDD-mediated CYP1A1 gene expression.

Cytochrome P450 1A1 (CYP1A1) is induced by halogenated and polycyclic aromatic hydrocarbons following activation of the aryl hydrocarbon receptor (AhR). Protein kinase C (PKC) has been implicated in the regulation of this response. In tissue culture, induction of PKC activity with phorbol esters synergizes the actions of TCDD-induced CYP1A1, while PKC inhibitors block induction of CYP1A1 by TCDD. Here, the actions of specific PKC inhibitors on CYP1A1 induction were examined using a HepG2 human cell line (TV101L) that carries a stably integrated firefly luciferase gene under control of the human CYP1A1 promoter (-1612/+293). TV101 cells were treated with TCDD and either the kinase inhibitor staurosporine or one of the PKC inhibitors GF109203X, G?6983, or G?6976. Aryl hydrocarbon receptor-dependent activation of CYP1A1-luciferase and cellular PKC activity were measured. TCDD treatment induced CYP1A1-luciferase activity in an AhR-dependent manner, as determined by binding of nuclear AhR to xenobiotic response elements (XREs). Dose-dependent inhibition of PKC activity by staurosporine was concordant with inhibition of TCDD-induced CYP1A1-luciferase activity. However, the PKC inhibitors GF109203X, G?6983, and G?6976 blocked PKC activity at concentrations independent of those necessary to block TCDD induction of CYP1A1-luciferase activity. For all inhibitors, reduction in CYP1A1-luciferase activity was independent of AhR activation, as determined by electrophoretic mobility shift analysis of TCDD-activated nuclear AhR. The specific PKC inhibitors did not significantly alter cytosolic or nuclear levels of AhR protein, whether alone or in combination with TCDD. These results suggested that PKC was not the sole factor responsible for regulation of CYP1A1.     

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.     

Protopine and allocryptopine increase mRNA levels of cytochromes P450 1A in human hepatocytes and HepG2 cells independently of AhR

The isoquinoline alkaloids protopine and allocryptopine are present in phytopreparations from medicinal plants, such as Fumaria officinalis. Since nothing is known about effects of the alkaloids on the expression of xenobiotic-metabolizing enzymes, we examined whether protopine or allocryptopine affect the expression of cytochromes P450 (CYPs) 1A1 and 1A2 in primary cultures of human hepatocytes and human hepatoma HepG2 cells. In HepG2 cells, protopine and allocryptopine significantly increased CYP1A1 mRNA levels after 24 h exposure at concentrations from 25 and 10 μM, respectively, as shown by real-time PCR. Both protopine and allocryptopine also dose-dependently increased CYP1A1 and CYP1A2 mRNA levels in human hepatocytes. However, the effects of the tested alkaloids on both cell models were much lower than the effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), a prototypical CYP1A inducer. Using gene reporter assays performed in transiently transfected HepG2 cells, we demonstrated that the induction of CYP1A1 expression by either protopine or allocryptopine was associated with mild or negligible activation of the aryl hydrocarbon receptor. In contrast to TCDD, CYP1A mRNA levels induced by protopine or allocryptopine in both HepG2 cells and human hepatocytes did not result in elevated CYP1A protein or activity levels as shown by western blotting and EROD assays, respectively. We conclude that the use of products containing protopine and/or allocryptopine may be considered safe in terms of possible induction of CYP1A enzymes.     

Effects of polybrominated diphenyl ethers on basal and TCDD-induced ethoxyresorufin activity and cytochrome P450-1A1 expression in MCF-7, HepG2, and H4IIE cells.

Polybrominated diphenylethers (PBDEs) are used as additive flame-retardants in consumer products to reduce the chances of ignition and burning. Levels of certain PBDE congeners have been increasing in fish, wildlife, and human tissues during the last decades. Some PBDEs are lipophilic and persistent, resulting in bioaccumulation in the environment. The structural similarity of PBDEs to other polyhalogenated aromatic hydrocarbons such as PCBs, has raised concerns that PBDEs might act as agonists for the aryl hydrocarbon receptor (AhR). To study the possible AhR-mediated effects of the environmentally relevant PBDEs (BDE47, 77, 99, 100, 153, 154, 183, 209), the induction of cytochrome P450-1A1 (CYP1A1) was studied in human breast carcinoma (MCF-7), human hepatocellular carcinoma (HepG2), and rat hepatoma (H4IIE) cells. 7-Ethoxyresorufin-O-deethylase (EROD) was used as a marker for CYP1A1 activity. Cells were exposed for 72 h to various PBDE concentrations (0.01-10 microM). Positive controls were 2,3,7,8-TCDD (0.001-2.5 nM) and PCB126 (0.01-10 nM). None of these PBDEs was capable of inducing EROD activity; this was confirmed by real time RT-PCR for CYP1A1 mRNA. However, in cells exposed to PBDEs in combination with TCDD, a concentration-dependent decrease in TCDD-induced EROD activity occurred. Co-exposure of BDE153 (10 muM) and a maximally inducing concentration of TCDD (1 nM) reduced EROD activity to 49% of the maximum induction by TCDD alone. All tested PBDEs showed similar effects in each cell line, though quantitative differences were observed. The observed decrease in CYP1A1 activity was not due to PBDE-dependent catalytic inhibition of EROD activity or cytotoxicity, nor were decreased CYP1A1 mRNA levels observed. However, inhibition of luciferase induction in mouse (Hepa) and rat (H4IIE) hepatoma cells containing a stably transfected AhR-responsive luciferase reporter gene, suggests that BDE77 is a weak AhR antagonist or partial agonist.     

Inhibition of heme oxygenase-1 partially reverses the arsenite-mediated decrease of CYP1A1, CYP1A2, CYP3A23, and CYP3A2 catalytic activity in isolated rat hepatocytes

Heme oxygenase (HO-1), the rate-limiting enzyme in the physiological breakdown of heme, is ubiquitous, and its expression can be increased by arsenite [As(III)], and similar other stimuli that induce cellular oxidative stress. Interestingly, it has been shown that the As(III)-induced HO-1 is inversely correlated with a decrease in cytochromes P450 (P450s) activity; however, the direct role for HO-1 in the inhibition of P450 enzymes remains unknown. Our results showed that As(III) at a concentration of 5 μM decreased the constitutive and inducible expression of CYP1A1, CYP1A2, CYP3A23, and CYP3A2 at the mRNA, protein, and catalytic activity levels. Moreover, As(III) decreased the nuclear accumulation of aryl hydrocarbon receptor (AhR) and pregnane X receptor without increasing their degradation. As(III) also increased the binding of cytosolic AhR to heat shock protein 90 and hepatitis B virus X-associated protein 2. In the presence of 2,3,7,8-tetrachlorodibenzo-p-dioxin as an inducer for CYP1A and rifampin as an inducer for CYP3A, As(III) decreased the enzymatic activity of the four P450s more than it decreased their mRNA or protein expression levels. It is noteworthy that treatment with the competitive HO-1 inhibitor, tin-mesoporphyrin, or supplementing external heme partially reversed the As(III)-mediated decrease in activities of the four P450s. In conclusion, the current study provides the first evidence that As(III) decreases CYP1A1, CYP1A2, CYP3A23, and CYP3A2 expression in freshly isolated rat primary hepatocytes. Furthermore, inhibiting the As(III)-mediated induction of HO-1 partially restores the enzymatic activity of these P450s that was initially decreased by As(III), confirming the direct role of HO-1 in the inhibition of P450s.     

Preferential induction of CYP1A1 and CYP1B1 in CCSP-positive cells.

Both benzo[a]pyrene (BaP) and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) are potent ligands of aryl hydrocarbon receptors (AhR). Although animal studies indicate that both compounds induce pathological changes in the peripheral lung, the specific cell type involved remains unclear. Clara cells, expressing Clara cell specific protein (CCSP) and abundant in cytochrome P450, are nonciliated bronchiolar epithelial cells in the peripheral lung. Here we explore the hypothesis that CCSP-positive Clara cells are highly responsive to AhR ligands and are the primary cell type involved in BaP- and TCDD-induced toxicities. The responsiveness to AhR ligands was evaluated by measuring the respective mRNA and protein levels of cytochrome P450 1A1 (CYP1A1) and 1B1 (CYP1B1) using real-time RT-PCR and immunocytochemistry assays. Two in vitro models were used: primary cultures of human small airway epithelial (SAE) cells and rat lung slice cultures. In the presence of calcium, human SAE cells differentiated into CCSP-positive cells. BaP- and TCDD-induced mRNA and protein levels of CYP1A1 and CYP1B1 levels were significantly elevated in CCSP-positive cell cultures. Similarly, AhR mRNA and protein levels were increased in CCSP-positive cell cultures, as determined by real-time RT-PCR and Western blot analysis. When rat lung slice cultures were treated with BaP or TCDD for 24 h, CYP1A1 and CYP1B1 proteins were strongly induced in Clara cells. These results indicate that, in the peripheral lung of both rats and humans, CCSP-positive cells (Clara cells) may be more sensitive to AhR ligands than other cell types.     

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.