TCDD-induced Expression Of CYP1A1 Research Articles

Negative regulation of aryl hydrocarbon receptor by its lysine mutations and exposure to nickel

Aryl Hydrocarbon Receptor (AhR) is a nuclear receptor for many environmental toxicants including TCDD. It mediates the induction of many metabolic enzymes of the cytochrome P450 family after engaging with its ligands. Meanwhile, nickel, also an environmental toxicant, induces a hypoxic response, leading to stabilization of HIF-1α. Lysine residues known for posttranslational modifications are important for subcellular localization, stability, and/or activity of gene products. In this study, we have determined nickel’s suppressive effect on TCDD-induced expression of CYP1A1 and CYP1A2, two members of the cytochrome P450 super-family. We have also analyzed CYP1A1 and CYP1A2 levels after expression of AhR with various lysine mutations. We have shown that induction of CYP1A1 and CYP1A2 by TCDD and other AhR ligands is significantly suppressed by hypoxia-mimetics including nickel. This effect is likely due to a competition for α-sub-unit shared by AhR and HIF-1 transcription factors. We have also found that substitutions of lysines 14, 17, and 21 with arginines suppress the ability of AhR to transactivate P450 genes after treatment with TCDD. Ligand-mediated AhR activation can be significantly modulated by co-exposure to other environmental toxicant (s) or by its lysine mutations.

The clock genes period 1 and period 2 mediate diurnal rhythms in dioxin-induced Cyp1A1 expression in the mouse mammary gland and liver

Transcription factors expressing Per-Arnt-Sim (PAS) domains are key components of the mammalian circadian clockworks found in most cells and tissues. Because these transcription factors interact with other PAS genes mediating xenobiotic metabolism and because toxin responses are often marked by daily variation, we determined whether the toxin-mediated activation of the signaling pathway involving several PAS genes, the aryl hydrocarbon receptor (AhR) and AhR nuclear translocator (ARNT), fluctuates rhythmically and whether this diurnal oscillation is affected by targeted disruption of key PAS genes in the circadian clockworks, Period 1 (Per1) and Per2. Treatment with the prototypical Ahr ligand, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), had inductive effects on a key target of AhR signaling, Cyp1A1, in both the mammary gland and liver of all animals. In wild type mice, the amplitude of this TCDD-induced Cyp1A1 expression in the mammary gland and liver was significantly greater (23–43-fold) during the night than during the daytime. However, the diurnal variation in the TCDD induction of mammary gland and liver Cyp1A1 expression was abolished in Per1 ldc, Per2 ldc and Per1 ldc/Per2 ldc mutant mice, suggesting that Per1, Per2 and their timekeeping function in the circadian clockworks mediate the diurnal modulation of AhR-regulated responses to TCDD in the mammary gland and liver.

Aryl hydrocarbon receptor pathway activation enhances gastric cancer cell invasiveness likely through a c-Jun-dependent induction of matrix metalloproteinase-9

BackgroundAbberant aryl hydrocarbon receptor (AhR) expression and AhR pathway activation are involved in gastric carcinogenesis. However, the relationship between AhR pathway activation and gastric cancer progression is still unclear. In present study, we used 2,3,7,8-tetrachlorodibenzo-para-dioxin (TCDD), a classic and most potent ligand of AhR, to activate AhR pathway and investigated the effect of AhR pathway activation on human gastric cancer AGS cell invasion and explored the corresponding mechanism.ResultsTo determine whether AhR pathway can be activated in AGS cells, we examined the expression of CYP1A1, a classic target gene of AhR pathway, following TCDD exposure. RT-PCR and western blot analysis showed that both CYP1A1 mRNA and protein expression were increased in a dose-dependent manner following TCDD treatment and AhR antagonist resveratrol (RSV) could reverse this TCDD-induced CYP1A1 expression. To determine whether TCDD treatment of AGS cells results in an induction of MMP-9 expression, we detected MMP-9 mRNA using RT-PCR and detected MMP-9 enzymatic activity using gelatin zymography. The results showed that both MMP-9 mRNA expression and enzymatic activity were gradually increased with the concentration increase of TCDD in media and these changes could be reversed by RSV treatment in a dose-dependent manner. To examine whether AhR activation-induced MMP-9 expression and activity in AGS cells results in increased migration and invasion, we performed wound healing migration assay and transwell migration and invasion assay. After TCDD treatment, the migration distance and the migration and invasion abilities of AGS cells were increased with a dose-dependent manner. To demonstrate AhR activation-induced MMP-9 expression is mediated by c-Jun, siRNA transfection was performed to silence c-Jun mRNA in AGS cells. The results showed that MMP-9 mRNA expression and activity in untreated control AGS cells were very weak; After TCDD (10 nmol/L) treatment, MMP-9 mRNA expression and activity were significant increased; This TCDD-induced MMP-9 expression and activity increase could be abolished by c-Jun siRNA transfection.ConclusionAhR pathway activation enhances gastric cancer cell invasiveness likely through a c-Jun-dependent induction of MMP-9. Our results provide insight into the mechanism and function of the AhR pathway and its impact on gastric cancer progression.

Disruption of period gene expression alters the inductive effects of dioxin on the AhR signaling pathway in the mouse liver

The aryl hydrocarbon receptor (AhR) and AhR nuclear translocator (ARNT) are transcription factors that express Per-Arnt-Sim (PAS) DNA-binding motifs and mediate the metabolism of drugs and environmental toxins in the liver. Because these transcription factors interact with other PAS genes in molecular feedback loops forming the mammalian circadian clockworks, we determined whether targeted disruption or siRNA inhibition of Per1 and Per2 expression alters toxin-mediated regulation of the AhR signaling pathway in the mouse liver and Hepa1c1c7 hepatoma cells in vitro. Treatment with the prototypical Ahr ligand, 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD), had inductive effects on the primary targets of AhR signaling, Cyp1A1 and Cyp1B1, in the liver of all animals, but genotype-based differences were evident such that the toxin-mediated induction of Cyp1A1 expression was significantly greater (2-fold) in mice with targeted disruption of Per1 ( Per1 ldc and Per1 ldc / Per2 ldc ). In vitro experiments yielded similar results demonstrating that siRNA inhibition of Per1 significantly increases the TCDD-induced expression of Cyp1A1 and Cyp1B1 in Hepa1c1c7 cells. Per2 inhibition in siRNA-infected Hepa1c1c7 cells had the opposite effect and significantly decreased both the induction of these p450 genes as well as AhR and Arnt expression in response to TCDD treatment. These findings suggest that Per1 may play a distinctive role in modulating AhR-regulated responses to TCDD in the liver.

TCDD-Induced CYP1A1 Expression, an Index of Dioxin Toxicity, Is Suppressed by Flavonoids Permeating the Human Intestinal Caco-2 Cell Monolayers

Since the toxicological effects of dioxins are mainly mediated by the aryl hydrocarbon receptor (AhR), an in vitro assessment system for AhR activity was used in this study to search for flavonoids that attenuated dioxin toxicity through the intestinal epithelial monolayer. When AhR transformation in Hepa-1c1c7 cells was examined by southwestern ELISA, nine flavonoids among 34 kinds of flavonoids inhibited the transformation by more than one-half. When each flavonoid with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) was added to dioxin-responsive HepG2 cells, seven flavonoids significantly restrained the TCDD-induced transcriptional activity of the CYP1A1 promoter. Furthermore, those seven flavonoids that had permeated the Caco-2 cell monolayers demonstrated an inhibitory effect on both the AhR transformation and on the transcriptional activity of the CYP1A1 promoter. The expression level of the CYP1A1 mRNA and protein induced by TCDD was suppressed by flavone, galangin, and tangeretin. It is proposed from these results that some flavonoids have the ability to suppress dioxin-induced AhR activity after permeating the human intestinal epithelial cell monolayer.

Comparison of the 2,3,7,8-tetrachlorodibenzo- p-dioxin (TCDD)-induced CYP1A1 gene expression profile in lymphocytes from mice, rats, and humans: Most potent induction in humans

2,3,7,8-Tetrachlorodibenzo- p-dioxin (TCDD) exerts its toxicity by binding a transcription factor, the aryl hydrocarbon receptor (AhR). C57BL/6 (C57) mice express AhRs that have high affinity for TCDD, and they strongly express target genes and develop severe toxic effects upon TCDD exposure. By contrast, DBA/2 (DBA) mice have a low-affinity form of AhR, weakly express target genes, and are resistant to TCDD. Although humans express low-affinity AhRs and have been assumed to be refractory to TCDD, their sensitivity to TCDD has yet to be determined. In this study we compared the TCDD-induced CYP1A1 gene expression profiles in lymphocytes from humans, C57 mice, DBA mice, and SD rats to obtain data as a basis for estimating human sensitivity to TCDD. Lymphocyte fractions prepared from the blood of individual humans and animals were cultured with TCDD. Their mRNAs for CYP1A1 and housekeeping genes were measured by RT-PCR or real-time PCR with primers designed for regions that are 100% homologous among each of the genes of all species/strains tested to obtain similar PCR efficiency. TCDD-induced CYP1A1 expression peaked at 2 h in DBA mice and SD rats and at 6 h in C57 mice and humans. At the peak times human lymphocytes showed the most potent CYP1A1 mRNA induction of the four species/strains tested. These results suggest that human lymphocytes are more sensitive to TCDD than the lymphocytes of mice and rats. Since the AhR-dependent gene expression did not reflect the AhR affinity for TCDD, these results also suggest that AhR-dependent gene expression in lymphocytes is modulated by an as yet unidentified mechanism in addition to the AhR affinity.

Inhibition of 2,3,7,8-tetrachlorodibenzo- p-dioxin (TCDD)-stimulated Cyp1a1 promoter activity by hypoxic agents

Since hypoxia-inducible factor-1α (HIF-1α) and the arylhydrocarbon receptor (AhR) shared the AhR nuclear translocator (Arnt) for hypoxia- and AhR-mediated signaling, respectively, it was possible to establish the hypothesis that hypoxia could regulate cytochrome P450 1a1 ( Cyp1a1) expression. In order to test this hypothesis, we undertook to examine the effect of hypoxia on Cyp1a1 transcription in Hepa-I cells. Mouse Cyp1a1 5′-flanking DNA, 1.6 kb was cloned into pGL3 expression vector in order to construct pm Cyp1a1-Luc. Hepa-I cells were transfected with pm Cyp1a1-Luc and treated with 2,3,7,8-tetrachlorodibenzo- p-dioxin (TCDD) in the presence or absence of various hypoxic agents such as 1–100 μM cobalt chloride, 1–100 μM picolinic acid, and 1–100 μM desferrioxamine. Luciferase activity of the reporter gene was measured from pm Cyp1a1-Luc-transfected Hepa-I cell lysate which contains 2 μg total protein using luciferin as a substrate. Hypoxic agents such as cobalt chloride, picolinic acid, and desferrioxamine showed inhibition of luciferase activity that was induced by 1-nM TCDD treatment in a dose-and time-dependent manner. Concomitant treatment of 150 μM ferrous sulfate with 1–100 μM desferrioxamine or 1–100 μM picolinic acid recovered luciferase activity from that inhibited by hypoxic agents or induced by TCDD. These data demonstrated that iron-chelating and hypoxic agents inhibited dioxin-induced Cyp1a1 transcription in Hepa-I cells. Thus, we might suggest that hypoxia inhibits TCDD-induced Cyp1a1 expression due to the competition between HIF-1α and the AhR for the Arnt in Hepa-I cells.

Characterization of CYP1A1 and CYP1A3 gene expression in rainbow trout ( Oncorhynchus mykiss)

The rainbow trout CYP1A1 and CYP1A3 genes share 96% amino acid identity and have similar enzymatic activity. The expression of CYP1A1 and 1A3 genes was investigated in 2,3,7,8-tetrachlorodibenzo- p-dioxin (TCDD)-treated rainbow trout tissues, sac fry and cell lines. Both CYP1A1 and CYP1A3 were induced by TCDD in all the tissues examined. While CYP1A3 gene was expressed constitutively at higher levels than CYP1A1 in trout intestine, preferential expression of CYP1A1 occurred in trout liver, heart, kidney and trout sac fry. In rainbow trout gonad (RTG)-2 and rainbow trout hepatoma (RTH)-149 cell lines, CYP1A1 was constitutively expressed and induced by exposure to TCDD, but CYP1A3 message was not detected, even after TCDD treatment. Quantitative analysis of CYP1A genes expression in rainbow trout liver revealed that TCDD induced CYP1A1 expression more than 50-fold and CYP1A3 RNA levels increased at least 100-fold over untreated fish. The cell- and tissue-specific expression indicates that these closely related CYP1A genes are independently regulated and that negative regulation may play a role in CYP1A3 gene expression.

Diflubenzuron, a Benzoyl-Urea Insecticide, Is a Potent Inhibitor of TCDD-Induced CYP1A1 Expression in HepG2 Cells

Diflubenzuron (DFB) belongs to a group of compounds called benzoyphenyl ureas acting as chitin synthesis inhibitors, which also inhibit growth of B16 murine melanomas. The present study was designed to investigate the effect of this insecticide, on CYP1A1 expression and induction in human hepatoma cells HepG2. Treatment of HepG2 cells over 72 h with noncytotoxic concentrations of DFB resulted in a strong dose-dependent decrease in constitutive ethoxyresorufin-O-deethylase activity. Moreover, DFB significantly decreased CYP1A1 induction by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) after 24 h exposure, as demonstrated by ethoxyresorufin-O-deethylase (EROD) activity and Northern blot analysis. Additional studies were performed both on parental HepG2 cells and HepG2–241c.1, which were stably transfected with the chloramphenicol acetyltransferase (CAT) reporter gene, cloned under the control of the human CYP1A1 promoter (−1140 to +59). Ribonuclease protection assays (RPA) analysis clearly demonstrated an inhibition of CYP1A1 transcription in both cell lines. Surprisingly, in corresponding experiments using 3-methylcholanthrene (3-MC) as a CYP1A1 inducer, DFB was less effective. Finally, in competitive binding studies using a 9S-enriched fraction of HepG2 cytosol, DFB was capable of displacing [3H]-2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) from its Ah receptor binding site. Taken together, these results support the involvement of a transcriptional mechanism in the inhibition of CYP1A1 expression in HepG2 cells by DFB, possibly via an Ah receptor antagonism.

Antioxidant response element-mediated 2,3,7,8-tetrachlorodibenzo- p-dioxin (TCDD) induction of human NAD(P)H:Quinone oxidoreductase 1 gene expression

Antioxidant response element (ARE) is required for high basal expression of the human NAD(P)H:quinone oxidoreductase 1 ( NQO1) gene in tumor cells and its induction in response to β-naphthoflavone and phenolic antioxidants. In this study, we have demonstrated that ARE also is required for induction of human NQO1 gene expression in response to 2,3,7,8-tetrachlorodibenzo- p-dioxin (TCDD). The various results suggest an alternate pathway for TCDD induction of human NQO1 gene expression. This pathway is independent of xenobiotic response element (XRE) and aromatic hydrocarbon (Ah) receptor. It is presumed that TCDD-induced expression of CYP1A1 leads to increased oxidative stress, resulting in transcriptional activation and/or modification of ARE-binding factors and increased expression of the human NQO1 gene.

Time- and Concentration-Dependent Induction of CYP1A1 and CYP1A2 in Precision-Cut Rat Liver Slices Incubated in Dynamic Organ Culture in the Presence of 2,3,7,8-Tetrachlorodibenzo-p-Dioxin

In a previous 24-h study, precision-cut rat liver slices were validated as a usefulin vitromodel for assessing the dose-related induction of CYP1A1 and CYP1A2 in rat liver following exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Further assessment of the utility of this model was accomplished by initially exposing rat liver slices to medium containing TCDD (0.01 nM) for 24 h and incubating the slices up to an additional 72 h in TCDD-free medium. The slices remained viable throughout the incubation period with an intracellular potassium content varying from 45.2 ± 2.3 μmol/g at 48 h to 50.0 ± 1.6 μmol/g at 72 h. In TCDD-exposed slices, CYP1A1 protein and its respective enzymatic activity, theO-deethylation of ethoxyresorufin (EROD), significantly increased with time over the 96-h incubation period, with EROD activity increasing from 63.6 ± 14.2 at 24 h to 905 ± 291 pmol/mg/min at 96 h. Under identical incubation conditions, but in the absence of TCDD, the EROD activity for the control liver slices ranged from 14.3 ± 4.3 to 44.9 ± 11.9 pmol/min/mg. Conversely, the level of CYP1A2 protein and its respective activity (acetanilide hydroxylation) transiently decreased from 24 to 96 h with no significant differences observed between the control (0 nM TCDD) and treatment group (0.01 nM TCDD). The concentration–effect relationship at 96 h was characterized by incubating rat liver slices for the initial 24 h in medium containing TCDD at concentrations ranging from 0.1 pM to 10 nM. Induction of CYP1A1 protein and EROD activity was observed for all treatment groups with the 10 nM TCDD treatment group displaying greater than 100-fold induction compared to control (0 nM TCDD). Immunohistochemical localization of CYP1A1 protein within liver slices supported the time- and concentration-dependent induction of EROD activity by TCDD. The induction of CYP1A1 was initially observed to be centrilobular, with increased expression due to both elevated CYP1A1 within cells and the recruitment of additional cells expressing CYP1A1 throughout the entire liver slice. Additionally, the immunohistochemical analysis of the liver slices demonstrated the conservation of tissue architecture following up to 96 h of incubation in dynamic organ culture and provided further evidence for maintenance of tissue viability. In comparison to CYP1A1, the induction of CYP1A2 at 96 h was a less sensitive response, with significant induction of CYP1A2 protein and its respective activity occurring at a medium concentration of 0.1 nM TCDD (686 pg/g liver). In general, increasing the incubation period from 24 to 96 h markedly increased TCDD-induced expression of CYP1A1 and minimally enhanced CYP1A2 expression. Moreover, extending the incubation period to 96 h resulted inin vitroinduction profiles for CYP1A1 and CYP1A2 that were qualitatively and quantitatively similar to that previously observed followingin vivoexposure to TCDD (Drahushuket al., Toxicol. Appl. Pharmacol.140, 393–403, 1996).

Effect of Transforming Growth Factor-β1on Expression of Aryl Hydrocarbon Receptor and Genes ofAhGene Battery: Clues for Independent Down-Regulation in A549 Cells

An inhibitory effect on both constitutive and inducible expression of cytochrome P450 isoenzymes has been shown for different cytokines and growth factors. We previously described an inhibition of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-induced CYP1A1 mRNA and enzyme activity by transforming growth factor-beta1 (TGF-beta1) in human lung cancer A549 cells. In the present study, we report that not only TCDD-induced expression of CYP1A1 but also basal mRNA expression of CYP1A1, CYP1B1, and aryl hydrocarbon receptor (AHR) was down-regulated by TGF-beta1 in cells not treated with TCDD. In contrast, mRNA expression of the AHR partner protein Arnt (aryl hydrocarbon receptor nuclear translocator) was not influenced. Furthermore, TCDD-induced expression of CYP1B1 and NMO-1 was inhibited, and the IC50 values of 5-10 pM TGF-beta1 were in the same range as observed for inhibition of CYP1A1 and AHR mRNA expression. Transfection studies with a plasmid containing a luciferase reporter gene under control of two dioxin-responsive elements indicate an effect on AHR protein expression. Results of time-course studies revealed a parallel inhibition of AHR and CYP1 mRNA expression, indicating that TGF-beta1 is a direct negative regulator of transcription of these genes. The treatment of cells with cycloheximide led to a superinduction of TCDD-induced CYP1A1 and CYP1B1 mRNA expression and abolished the inhibitory effect of TGF-beta1 on basal as well as TCDD-induced CYP1 and AHR mRNA expression. TGF-beta1 seems not to influence the stability of AHR mRNA. The results suggest that TGF-beta1 induces rapid transcription and translation of an as-yet-unknown negative regulatory factor or factors that may directly regulate expression of AHR and genes of Ah gene battery.

Potentiation of CYP1A1 Gene Expression in MCF-7 Human Breast Cancer Cells Cotreated with 2,3,7,8-Tetrachlorodibenzo- p-Dioxin and 12- O-Tetradecanoylphorbol-13-Acetate

In MCF-7 cells treated with 2,3,7,8-tetrachiorodibenzo- p-dioxin (TCDD), 12- O-tetradecanoylphorbol-13-acetate (TPA) causes a time- and concentration-dependent modulation of TCDD-induced CYP1A1 gene expression. Treatment of MCF-7 cells with 1 nM TCDD for 24 h resulted in the induction of ethoxyresorufin O-deethylase (EROD) activity and CYP1A1 mRNA levels. In cells treated with TCDD for 24 h and 100 ng/ml TPA for 26 and 30 h, the TCDD-induced CYP1A1 gene expression was decreased. For example, TCDD-induced EROD activity decreased from 122 pmol/min/mg to 25.5 pmol/min/mg after treatment of MCF-7 cells with TPA for 26 h and this was also paralleled by a 44% decrease in CYP1A1 mRNA levels. There was also a decrease in nuclear Ah receptor levels and the binding of nuclear extracts to a 32P-labeled dioxin responsive element (DRE) in a gel mobility shift assay. In parallel studies which measured EROD activities, similar TCDD/TPA interactions were observed in wild-type Hepa 1c1c7 cells, whereas no interactive effects were observed in T47-D human breast cancer cells. In MCF-7 cells treated with TPA for 36 or 48 h, the TCDD-induced EROD activity and CYP1A1 mRNA levels were restored and in cells exposed to TPA for 72 or 96 h superinducibility of CYP1Al gene expression was observed; there was a 2.8-and 2.2-fold increase in EROD activity and CYP1A1 mRNA levels, respectively, compared to MCF-7 cells treated with TCDD alone. The biphasic temporal effects of TPA on TCDD-induced CYP1A1 gene expression in MCF-7 cells were paralleled by comparable changes in nuclear Ah receptor levels and binding to a synthetic DRE. In contrast, prolonged exposure of the wild-type Hepa 1c1c7 or T47-D cells to both TCDD plus TPA gave results similar to those observed after 24 h. These data show that the effects of TPA on TCDD-induced expression of CYP1A1 are cell-specific and suggest that the proposed protein kinase C (PKC)-dependent activation of the nuclear Ah receptor complex may not be required in MCF-7 cells since TPA downregulates PKC activity within 11 h and this inactivation persists for at least 96 h.