Risk of bleeding and stroke with oral anticoagulation and antiplatelet therapy in patients with atrial fibrillation in Taiwan: a nationwide cohort study.

BACKGROUND: Data on the use of oral anticoagulation (OAC) and antiplatelet therapy and the risk of bleeding and stroke amongst Asian patients with atrial fibrillation (AF) are limited. We investigated the risks of bleeding and stroke with use of oral anticoagulation (OAC) and antiplatelet therapy as mono- or combination therapy, in patients with AF from a Chinese nationwide cohort study. METHODS: We studied a cohort of 10384 patients (57.2% men, age 67.8 ± 13.2 yrs) between 1999 and 2010 from the National Health Insurance Research Database in Taiwan. Records of prescriptions were obtained during follow-up. The main outcome was a recurrent stroke during the follow-up period. Time-dependent Cox proportional hazards models were used for this analysis. RESULTS: We documented 1009 events for bleeding, as well as 224 hemorrhagic stroke and 1642 ischemic stroke events during a median 3.2 (interquartile range, 1.05-6.54) years' follow-up. Compared with warfarin users, patients with antiplatelet therapy had a lower risk of bleeding (adjusted relative risk [RR], 0.59, 95% confidence interval [CI], 0.49-0.71, p<0.001) whilst combination therapy had a non-statistically significant higher bleeding risk (RR, 1.33, 95%, 0.91-1.94, p = 0.20). Patients on antiplatelet monotherapy had a similar risk for ischemic stroke compared with OAC (RR 1.05, 95% CI, 0.89-1.25, p = 0.50), whilst those on combination therapy had a significantly higher risk (RR 1.90, 95% CI, 1.34-2.70, p<0.001). CONCLUSION: In a national representative cohort, antiplatelet therapy had no significant difference in ischemic stroke risk to warfarin. For bleeding, aspirin had a lower risk compared to warfarin. This may reflect poor anticoagulation control, highlighting important missed opportunities for improved stroke prevention, especially in countries where anticoagulation management is suboptimal.

Dithiolethione modified valproate and diclofenac increase E-cadherin expression and decrease proliferation of non-small cell lung cancer cells.

The effects of dithiolethione modified valproate, diclofenac and sulindac on non-small cell lung cancer (NSCLC) cells were investigated. Sulfur(S)-valproate and S-diclofenac at 1 microg/ml concentrations significantly reduced prostaglandin (PG)E(2) levels in NSCLC cell lines A549 and NCI-H1299 as did the COX-2 inhibitor DuP-697. In vitro, S-valproate, S-diclofenac and S-sulindac half-maximally inhibited the clonal growth of NCI-H1299 cells at 6, 6 and 15 microg/ml, respectively. Using the MTT assay, 10 microg/ml S-valproate, NO-aspirin and Cay10404, a selective COX-2 inhibitor, but not SC-560, a selective COX-1 inhibitor, inhibited the growth of A549 cells. In vivo, 18mg/kg i.p. of S-valproate and S-diclofenac, but not S-sulindac, significantly inhibited A549 or NCI-H1299 xenograft proliferation in nude mice, but had no effect on the nude mouse body weight. The mechanism by which S-valproate and S-diclofenac inhibited the growth of NSCLC cells was investigated. Nitric oxide-aspirin but not S-valproate caused apoptosis of NSCLC cells. By Western blot, S-valproate and S-diclofenac increased E-cadherin but reduced vimentin and ZEB1 (a transcriptional suppressor of E-cadherin) protein expression in NSCLC cells. Because S-valproate and S-diclofenac inhibit the growth of NSCLC cells and reduce PGE(2) levels, they may prove beneficial in the chemoprevention and/or therapy of NSCLC.

Modulation of carcinogen metabolism by nitric oxide-aspirin 2 is associated with suppression of DNA damage and DNA adduct formation.

Nitric oxide (NO)-donating non-steroidal anti-inflammatory drugs (NSAIDs) represent a promising new class of drugs developed to provide a safer alternative than their conventional NSAID counterparts in chemoprevention. We tested the effects of NO-aspirin 2 on Phase I and Phase II carcinogen-metabolizing enzymes. In HepG2 human hepatoma cells and in LS180 colonic adenocarcinoma cells, NO-aspirin 2 inhibited 2,3,7,8-tetrachlordibenzo-p-dioxin (TCDD)-induced cytochrome P450 (CYP) enzyme activity and CYP1A1 and CYP1A2 mRNA expression. These effects were further characterized as being mediated through transcriptional regulation: NO-aspirin 2 inhibited binding of ligand (TCDD)-activated aryl hydrocarbon receptor to the CYP1A1 enhancer sequence; additionally, NO-aspirin 2 suppressed carcinogen-induced expression of CYP1A heterogeneous nuclear RNA. The fate of carcinogen metabolites depends not only on activation by CYP enzymes but also detoxification by Phase II enzymes. Both HepG2 and LS180 cells treated with NO-aspirin 2 showed an increase in glutathione S-transferase-P1 (GST-P1), glutamate-cysteine ligase (GCL), and NAD(P)H:quinone oxidoreductase-1 (NQO1) expression. Compared with two other NO-releasing compounds, diethylenetriamine-NO and the organic nitrate, isosorbide dinitrate, the inhibitory effects of NO-aspirin 2 on TCDD-induced CYP activity and mRNA expression were considerably more potent. Furthermore, aspirin alone had no inhibitory effect on TCDD-induced CYP activity, nor did aspirin up-regulate GCL, GST-P1, or NQO1 expression. Consequent to the effects on carcinogen-metabolizing enzymes, NO-aspirin 2 inhibited [3H]benzo[a]pyrene-DNA adduct formation and DNA damage elicited by TCDD or benzo[a]pyrene. Our results demonstrate that NO-aspirin 2 may be an effective chemopreventive agent by favorably affecting the inhibitory and enhancing effects of Phase I and Phase II carcinogen metabolism, thereby protecting DNA from carcinogenic insult.

Novel dithiolethione-modified nonsteroidal anti-inflammatory drugs in human hepatoma HepG2 and colon LS180 cells.

PURPOSE: Nonsteroidal anti-inflammatory drugs (NSAID) are promising chemopreventive agents against colon and other cancers. However, the molecular basis mediated by NSAIDs for chemoprevention has not been fully elucidated. Environmental carcinogens induce DNA mutation and cellular transformation; therefore, we examined the effect of NSAIDs on carcinogenesis mediated by the aryl hydrocarbon receptor signaling pathway. In this study, we investigated the activities of a new class of NSAIDs containing dithiolethione moieties (S-NSAID) on both arms of carcinogenesis. EXPERIMENTAL DESIGN: We investigated the effects of the S-NSAIDs, S-diclofenac and S-sulindac, on carcinogen activation and detoxification mechanisms in human hepatoma HepG2 and human colonic adenocarcinoma LS180 cells. RESULTS: We found that S-diclofenac and S-sulindac inhibited the activity and expression of the carcinogen activating enzymes, cytochromes P-450 (CYP) CYP1A1, CYP1B1, and CYP1A2. Inhibition was mediated by transcriptional regulation of the aryl hydrocarbon receptor (AhR) pathway. The S-NSAIDs down-regulated carcinogen-induced expression of CYP1A1 heterogeneous nuclear RNA, a measure of transcription rate. Both compounds blocked carcinogen-activated AhR from binding to the xenobiotic responsive element as shown by chromatin immunoprecipitation. S-diclofenac and S-sulindac inhibited carcinogen-induced CYP enzyme activity through direct inhibition as well as through decreased transcriptional activation of the AhR. S-sulindac induced expression of several carcinogen detoxification enzymes of the glutathione cycle including glutathione S-transferase A2, glutamate cysteine ligase catalytic subunit, glutamate cysteine ligase modifier subunit, and glutathione reductase. CONCLUSIONS: These results indicate that S-diclofenac and S-sulindac may serve as effective chemoprevention agents by favorably balancing the equation of carcinogen activation and detoxification mechanisms.

Generation of nitroxyl by heme protein-mediated peroxidation of hydroxylamine but not N-hydroxy-L-arginine.

The chemical reactivity, toxicology, and pharmacological responses to nitroxyl (HNO) are often distinctly different from those of nitric oxide (NO). The discovery that HNO donors may have pharmacological utility for treatment of cardiovascular disorders such as heart failure and ischemia reperfusion has led to increased speculation of potential endogenous pathways for HNO biosynthesis. Here, the ability of heme proteins to utilize H2O2 to oxidize hydroxylamine (NH2OH) or N-hydroxy-L-arginine (NOHA) to HNO was examined. Formation of HNO was evaluated with a recently developed selective assay in which the reaction products in the presence of reduced glutathione (GSH) were quantified by HPLC. Release of HNO from the heme pocket was indicated by formation of sulfinamide (GS(O)NH2), while the yields of nitrite and nitrate signified the degree of intramolecular recombination of HNO with the heme. Formation of GS(O)NH2 was observed upon oxidation of NH2OH, whereas NOHA, the primary intermediate in oxidation of L-arginine by NO synthase, was apparently resistant to oxidation by the heme proteins utilized. In the presence of NH2OH, the highest yields of GS(O)NH2 were observed with proteins in which the heme was coordinated to a histidine (horseradish peroxidase, lactoperoxidase, myeloperoxidase, myoglobin, and hemoglobin) in contrast to a tyrosine (catalase) or cysteine (cytochrome P450). That peroxidation of NH2OH by horseradish peroxidase produced free HNO, which was able to affect intracellular targets, was verified by conversion of 4,5-diaminofluorescein to the corresponding fluorophore within intact cells.

Sulindac and its metabolites induce carcinogen metabolizing enzymes in human colon cancer cells.

Sulindac is a nonsteroidal antiinflammatory drug that has been demonstrated to be a potent chemopreventive agent against colorectal cancer in both human and animal models. In vivo, sulindac may be reversibly reduced to the active antiinflammatory compound, sulindac sulfide, or irreversibly oxidized to sulindac sulfone. Sulindac has also been shown to inhibit polycyclic aromatic hydrocarbon (PAH)-induced cancer, but the molecular mechanisms of its antitumor effect remain unclear. In this study, we investigated the effects of sulindac and its metabolites on the expression of enzymes that metabolize and detoxify PAHs in 2 human colon cancer cell lines, LS180 and Caco-2. Sulindac and sulindac sulfide induced a sustained, concentration-dependent increase in CYP enzyme activity as well as an increase in the mRNA levels of CYP1A1, CYP1A2 and CYP1B1. Sulindac and sulindac sulfide induced the transcription of the CYP1A1 gene, as measured by the level of heterogeneous nuclear CYP1A1 RNA and verified by the use of actinomycin D as a transcription inhibitor. Chromatin immunoprecipitation assays demonstrated that sulindac and sulindac sulfide also increased the nuclear level of activated aryl hydrocarbon receptor, the transcription factor which mediates CYP expression. Additionally, sulindac and both metabolites increased the activity and mRNA expression of the carcinogen detoxification enzyme NAD(P)H:quinone oxidoreductase, as well as the expression of UDP-glucuronosyltransferase mRNA. These results show an overall upregulation of carcinogen metabolizing enzymes in colon cancer cells treated with sulindac, sulindac sulfide and sulindac sulfone that may contribute to the established chemoprotective effects of these compounds.

A novel synthetic analogue of a constituent of Isodon excisus inhibits transcription of CYP1A1, -1A2 and -1B1 by preventing activation of the aryl hydrocarbon receptor.

We investigated the effect of a novel synthetic analogue of a constituent from the Chinese medicinal herb Isodon excisus, 3-(3-methoxy-phenyl)-N-(3, 4, 5-trimethoxy-phenyl)-acrylamide (compound 343), on the carcinogen activation pathway mediated by the aryl hydrocarbon receptor (AhR) in human hepatoma HepG2 cells. We found that compound 343 inhibited the upregulation of cytochrome P-450 (CYP) enzyme activity in cells treated with the AhR ligands and potent carcinogens, dimethylbenz[a]anthracene (DMBA) or 2, 3, 7, 8-tetrachlorodibenzo-p-dioxin (TCDD). Compound 343 also inhibited the DMBA- or TCDD-induced increase in CYP1A1, -1A2 and -1B1 mRNA levels. Carcinogen-induced transcription of CYP genes was also suppressed by compound 343, as measured by a reporter gene controlled by the xenobiotic-responsive element (XRE). This was confirmed by measuring the amount of carcinogen-induced CYP1A1 heterogeneous nuclear RNA. Compound 343 blocked the DMBA- or TCDD-induced activation of the AhR DNA-binding capacity for the XRE, as measured by a chromatin immunoprecipitation assay. Compound 343 also inhibited CYP enzyme activity in microsomes isolated from DMBA- or TCDD-treated cells, as well as the activity of recombinant CYP1A1, -1A2 and -1B1, indicating that compound 343 directly inhibits CYP enzymes. These results indicate that compound 343 is both a potent inhibitor of carcinogen-induced CYP enzyme expression, as well as a direct inhibitor of CYP enzymes.

Sulindac regulates the aryl hydrocarbon receptor-mediated expression of Phase 1 metabolic enzymes in vivo and in vitro.

Sulindac, a widely used non-steroidal anti-inflammatory drug (NSAID), has been shown to inhibit chemically induced carcinogenesis in animal models. In the present study, we have investigated the molecular mechanism by which sulindac affects the activity and expression of the enzymes that mediate the initial detoxification steps of many environmental carcinogens, the cytochromes P450 1A1, 1A2 and 1B1. Sulindac treatment of Sprague-Dawley rats resulted in a dose-dependent increase in hepatic cytochrome P450 (CYP) enzyme activity and in the expression of hepatic CYPs 1A1 and 1B1 mRNA. In the HepG2 human liver cancer cell line, sulindac caused a sustained, dose-dependent increase in CYP enzyme activity. Sulindac treatment resulted in a profound, dose-dependent increase in CYP 1A1 mRNA and a modest increase in 1A2 mRNA. The increase in CYP 1A1 mRNA induced by sulindac was, like enzyme activity, sustained for several days after the initial treatment. Sulindac induced the transcription of the CYP1A1 gene, as measured by the level of heterogeneous nuclear 1A1 RNA and by actinomycin D chase experiment. Since the transcription of CYP1A1 is under the control of the aryl hydrocarbon receptor (AhR), we examined the ability of sulindac to activate the receptor. Sulindac bound to the AhR, as measured by ligand-binding assay, and induced the binding of the AhR with the xenobiotic-responsive element present in the promoter region of the CYP1A1 gene. These results are the first demonstration that NSAIDs modulate carcinogen metabolic enzymes and provide a novel mechanism to explain the established chemopreventive activity of sulindac.

The biology of tobacco and nicotine: bench to bedside.

Strong epidemiologic evidence links smoking and cancer. An increased understanding of the molecular biology of tobacco-related cancers could advance progress toward improving smoking cessation and patient management. Knowledge gaps between tobacco addiction, tumorigenesis, and cancer brought an interdisciplinary group of investigators together to discuss "The Biology of Nicotine and Tobacco: Bench to Bedside." Presentations on the signaling pathways and pathogenesis in tobacco-related cancers, mouse models of addiction, imaging and regulation of nicotinic receptors, the genetic basis for tobacco carcinogenesis and development of lung cancer, and molecular mechanisms of carcinogenesis were heard. Importantly, new opportunities to use molecular biology to identify and abrogate tobacco-mediated carcinogenesis and to identify high-risk individuals were recognized.

The antitumor drug candidate 2-(4-amino-3-methylphenyl)-5-fluorobenzothiazole induces NF-kappaB activity in drug-sensitive MCF-7 cells.

2-(4-Amino-3-methylphenyl)-5-fluoro-benzothiazole (5F 203) potently inhibits MCF-7 breast cancer cell growth in part by activating the aryl hydrocarbon receptor (AhR) signaling pathway. Ligands for the AhR (i.e. dioxin) have also been shown to modulate the NF-kappaB signaling cascade, affecting physiological processes such as cellular immunity, inflammation, proliferation and survival. The objective of this study was to investigate the effect of 5F 203 treatment on the NF-kappaB signaling pathway in breast cancer cells. Exposure of MCF-7 cells to 5F 203 increased protein-DNA complex formation on the NF-kappaB-responsive element as determined by electrophoretic mobility shift assay, but this effect was eliminated in MDA-MB-435 cells, which are resistant to the antiproliferative effects of 5F 203. An increase in NF-kappaB-dependent transcriptional activity was confirmed by a significant increase in NF-kappaB-dependent reporter activity in sensitive MCF-7 cells, which was absent in resistant MDA-MB-435 cells and AhR-deficient subclones of MCF-7 cells. Inhibition of NF-kappaB activation enhanced the increase in xenobiotic response element-dependent reporter activity in MCF-7 cells when treated with 5F 203. The drug candidate 5F 203 also induced mRNA levels of IL-6, an NF-kappaB-responsive gene, in MCF-7 cells, but not in MDA-MB-435 cells, as determined by quantitative RT-PCR. These findings suggest that 5F 203 activation of the NF-kappaB signaling cascade may contribute to 5F 203-mediated anticancer activity in human breast cancer MCF-7 cells.

Aryl hydrocarbon receptor activation of an antitumor aminoflavone: basis of selective toxicity for MCF-7 breast tumor cells.

Aminoflavone (4H-1-benzopyran-4-one, 5-amino-2-(4-amino-3-fluorophenyl)-6,8-difluoro-7-methyl; NSC 686288) demonstrates differential antiproliferative activity in the National Cancer Institute's anticancer drug screen. We demonstrate here that MCF-7 human breast cancer cells are sensitive to aminoflavone both in vitro and when grown in vivo as xenografts in athymic mice. As previous work has indicated that aminoflavone requires metabolic activation by cytochrome P450 1A1 (CYP1A1), we investigated the effect of aminoflavone on CYP1A1 expression and on the aryl hydrocarbon receptor (AhR), a transcriptional regulator of CYP1A1. In aminoflavone-sensitive but not aminoflavone-resistant cells, the drug caused a 100-fold induction of CYP1A1 mRNA and a corresponding increase in ethoxyresorufin-O-deethylase activity. An AhR-deficient variant of the MCF-7 breast carcinoma, AH(R100), with diminished CYP1A1 inducibility, exhibits cellular resistance to aminoflavone and is refractory to CYP1A1 mRNA induction by the drug. The increase in CYP1A1 mRNA in the aminoflavone-sensitive MCF-7 breast tumor cell results from transcriptional activation of xenobiotic-responsive element (XRE)-controlled transcription. Aminoflavone treatment causes a translocation of the AhR from the cytoplasm to the nucleus with subsequent formation of AhR-XRE protein DNA complexes. In contrast to the aminoflavone-sensitive MCF-7 cells, the resistant cell lines (MDA-MB-435, PC-3, and AH(R100)) demonstrated constitutive nuclear localization of AhR. Additionally, aminoflavone failed to induce ethoxyresorufin-O-deethylase activity, CYP1A1 transcription, AhR-XRE complex formation, and apoptosis in aminoflavone-resistant cells. These results suggest that the cytotoxicity of aminoflavone in a sensitive breast tumor cell line is the result of the engagement of AhR-mediated signal transduction.

The drug salicylamide is an antagonist of the aryl hydrocarbon receptor that inhibits signal transduction induced by 2,3,7,8-tetrachlorodibenzo-p-dioxin.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is a widespread environmental contaminant, that has been linked with a variety of deleterious effects on human health, including increased cancer rates and reproductive anomalies. The detrimental effects of TCDD are mediated via the aryl hydrocarbon receptor (AhR), a transcription factor that regulates the expression of the carcinogen-activating enzymes cytochromes P-450 (CYP) 1A1, 1A2, and 1B1. In the present study, we examined the ability of synthetic derivatives of salicylic acid to affect TCDD-stimulated AhR-mediated signal transduction in human hepatoma HepG2 cells. Salicylamide (SAL), an analgesic drug, caused a potent and long-lasting inhibition of TCDD-induced CYP enzyme activity. Acetylsalicylic acid (aspirin) and the naturally occurring phytochemical salicylic acid had no effect on CYP activity. SAL inhibited the increase in CYP1A1, -1A2, and -1B1 mRNA levels that occurs on exposure to TCDD. TCDD-induced transcription of these genes was also inhibited by SAL, but not by aspirin or salicylic acid, as demonstrated by luciferase reporter assays. The transcription of the CYP1 family of genes is regulated by the interaction of TCDD-activated AhR with the xenobiotic-responsive element present in the promoter regions of these genes. As shown by electrophoretic mobility shift assay, SAL completely blocked the binding of TCDD-activated AhR to the xenobiotic responsive element. Also, SAL substantially blocked the binding of TCDD to the cytosolic AhR. These results demonstrate that SAL, a commonly used analgesic, is a potent inhibitor of AhR-mediated signal transduction, and may be an effective agent in the prevention of TCDD-associated disease.

Dehydroepiandrosterone inhibits the expression of carcinogen-activating enzymes in vivo.

We investigated the effect of the steroid hormone dehydroepiandrosterone (DHEA) on the hepatic expression and activity of carcinogen-activating enzymes, the cytochromes P450 (CYP) 1A1, 1A2 and 1B1, in Sprague-Dawley rats. In animals fed DHEA at 200 or 400 mg/kg body weight every other day for 2 weeks prior to exposure to the aryl hydrocarbon dimethylbenz[a]anthracene (DMBA, 5 mg/kg), there was a dose-dependent decrease in hepatic CYP activity, as measured by ethoxyresorufin-O (EROD) assay, from 37.1 to 22.9 and 14.7 pmoles/min/10 microg microsomes, respectively. DHEA did not directly inhibit microsomal EROD activity, however, leading us to investigate its effects on enzyme expression. To test this, we examined protein and mRNA levels of the enzymes. Western blot for CYP1A1 and CYP1A2 showed that DHEA inhibited the increase in hepatic CYP1A1 and CYP1A2 enzyme levels that are normally induced by DMBA. DMBA-induced increase in expression of CYP1A1, CYP1A2 and CYP1B1 mRNA was similarly blunted in DHEA-treated animals. DHEA was also able to significantly reduce the basal expression of CYP1A1 and CYP1A2 but not of CYP1B1. These results indicate that DHEA regulates the expression and, hence, the activity of hepatic carcinogen-activating enzymes in vivo, and this may be an important mechanism of its chemopreventive activity.

Complement activation is responsible for acute toxicities in rhesus monkeys treated with a phosphorothioate oligodeoxynucleotide.

The objective of this study was to define the role of complement activation in the acute and transient toxicities associated with administration of phosphorothioate oligonucleotides in monkeys. In the absence of complement inhibitor, complement activation blocker-2 (CAB-2), i.v. infusion of 20 mg/kg ISIS 2302 produced increases in the concentrations of the complement split products Bb and C5a (100- and 7-fold, respectively). Monkeys also experienced marked changes in bloodpressure (hypertension and hypotension), clinical signs of toxicity (lethargy and periorbital edema), fluctuations in circulating neutrophil counts, and elevations in serum cytokine levels (45-, 12-, and 4-fold increases in IL-6, MCP-1, and IL-12, respectively). Changes occurred at or near the end of infusion and returned to normal over time. One of the three animals died approximately 4 h following infusion of 20 mg/kg ISIS 2302 alone. In contrast, prior treatment with CAB-2 effectively blocked complement activation, as well as the ISIS 2302-induced hemodynamic and clinical responses. Importantly, plasma concentration of ISIS 2302 were unaffected by CAB-2 pretreatment. Thus, the protection afforded by CAB-2 was due to its inhibition of complement activation rather than to any impact on the disposition of ISIS 2302. These results clearly demonstrate the causal relationship between activation of the alternative complement pathway and the hemodynamic and clinical responses associated with rapid infusion of phosphorothioate oligonucleotides. Demonstration of this relationship underscores the importance of avoiding complement activation in patients to ensure the continued safe use of phosphorothioate oligodeoxynucleotides.

Resistance of MCF-7 cells to dimethylbenz(a)anthracene-induced apoptosis is due to reduced CYP1A1 expression.

We have developed a series of aryl hydrocarbon (AH)-resistant cell lines derived from MCF-7 human breast epithelial cancer cells by continuous exposure to the AH benzo[a]pyrene. These cell lines display cross-resistance to the mammary carcinogen dimethylbenz[a]anthracene (DMBA). Apoptosis induced by exposure to DMBA is greatly decreased in the resistant cell lines compared to the wild-type, in proportion to the level of resistance. Apoptosis induced by DMBA could be blocked by inhibitors of DMBA metabolism such as alpha-naphthoflavone and diosmetin. We therefore examined the resistant cell lines for their ability to metabolize DMBA and for the formation of DMBA-DNA adducts, and found that both parameters were decreased compared to wild-type cells in proportion to the level of resistance. When exposed to DMBA or 2,3,7,8-tetrachlorodibenzo-p-dioxin, the resistant cell lines have a diminished capacity to carry out ethoxyresorufin-O-deethylation, indicating that the induction of cytochrome P450 1A1 (CYP1A1) enzyme is impaired. We therefore examined the expression of the CYP1A1 gene, and found reduced levels of both CYP1A1 mRNA and CYP1A1-promoter controlled transcription in resistant cells compared to the wild-type. The deleterious effects of AHs are believed to be mediated by the aryl hydrocarbon receptor (AhR), a ligand-activated transcription factor which regulates CYP1A1 expression. Resistant cell lines had a reduced expression of the AhR, as measured at the mRNA and protein levels. These data demonstrate that AH resistance in these cells is mediated by changes in the signal transduction pathway which regulates CYP1A1 expression.

Inhibition of carcinogen-activating enzymes by 16alpha-fluoro-5-androsten-17-one.

In the present study, we examined the effect of a synthetic analogue of the chemopreventive hormone dehydroepiandrosterone, 16alpha-fluoro-5-androsten-17-one, also known as fluasterone, on the activity and expression of carcinogen-activating enzymes in MCF-7 cells. The increase in cytochrome P450 (CYP) 1A1 and 1B1 activity, as measured by ethoxyresorufin-O-deethylase activity, in cells treated with the carcinogens dimethylbenzanthracene (DMBA) or 2,3,5,7-tetrachlorodibenzo-p-dioxin (TCDD), was inhibited by cotreatment with fluasterone. However, treatment of the cells with fluasterone after induction with DMBA or TCDD failed to decrease enzyme activity, indicating that inhibition was not the result of direct enzyme inhibition. Therefore, we examined the effect of fluasterone on gene expression at the mRNA level. Both DMBA and TCDD caused a dramatic increase in the amount of CYP1A1 and CYP1B1 mRNA, the two major isoforms involved in carcinogen activation in these cells. In cells cotreated with fluasterone, however, there was a dose-dependent decrease in CYP1A1 and CYP1B1 mRNA. Fluasterone also inhibited the basal level of CYP1A1 mRNA but not CYP1B1. Fluasterone inhibited the rate of CYP1A1 promoter-controlled transcription, indicating that it affects the transcriptional regulation of the gene. Actinomycin D chase experiments showed that fluasterone also caused an increase in the degradation of CYP1A1 mRNA, while leaving CYP1B1 mRNA unaffected. These results indicate that fluasterone inhibits the increase in the expression of CYP1A1 normally caused by exposure to carcinogens by both transcriptional and post-transcriptional mechanisms and that CYP1B1 expression is not susceptible to the same post-transcriptional mechanism.