Profiling of CYP4Z1 and CYP1B1 expression in bladder cancers.

Bladder cancer is the tenth most common cancer worldwide, where its burden remains a challenge and needs new novel therapies. Several reports indicate expression of CYP4Z1 and CYP1B1 in many tumours. Their expressions are associated with a poor prognosis, and therefore proposed as promising biomarkers or targets for anticancer therapy. By using immunohistochemistry, expression of CYP4Z1 and CYP1B1 was evaluated in a panel of different types of bladder cancer, and the enzymes' relation to histopathological features were assessed. Results showed an increased expression of CYP4Z1 (54.3%) and CYP1B1 (76.9%) in the majority of bladder cancers compared to weak or lack of expression of both enzymes in normal tissues. CYP4Z1expression was significantly associated with tumour grade and stage where the expression was markedly increased in a high grade and advanced stage of the disease (p < 0.05). Additionally, CYP1B1 expression was also associated with TNM staging (p < 0.05) and its expression was increased in patients with lymph node metastasis. The expression profiles of CYP4Z1 and CYP1B1 suggest that both enzymes have the potential to be biomarkers or targets for novel anticancer therapy for bladder cancer. Nevertheless, further studies are needed to better delineate whether these enzymes are druggable targets.

LncRNA H19 suppresses pyroptosis of cardiomyocytes to attenuate myocardial infarction in a PBX3/CYP1B1-dependent manner.

OBJECTIVE: Myocardial infarction (MI) is a major cause of cardiovascular disease which poses great healthy and financial burden for individuals. MI can be mainly induced by hypoxia. Therefore, in this study, we aimed to explore the function and mechanism of lncRNA H19 on hypoxia-induced pyroptosis of cardiomyocytes. METHOD: Peripheral blood from healthy controls and MI patients was collected for determination of mRNA and protein expression levels of H19 and CYP1B1. The correlation between these two factors was analyzed. Then MI rat model was established and injected with H19 overexpression/CYP1B1 knockdown plasmid, in which the infraction area and pathological morphology were observed. Hypoxic cardiomyocytes were transfected with overexpression or knockdown of H19 and CYP1B1 for determination of NLRP3, ASC, caspase-1, IL-1ß, IL-18, CyclinD1, and PCNA. Cell proliferation ability was assessed by CCK8. RIP and dual luciferase gene reporter assay were applied to verify the binding among H19, PBX3 and CYP1B1. RESULTS: Downregulated H19 and upregulated CYP1B1 were observed in MI patients. A negative correlation was found for H19 and CYP1B1 expressions. Transfection of H19 overexpression or CYP1B1 knockdown could attenuate the MI progression in MI rats. In hypoxic cardiomyocytes, H19 overexpression or CYP1B1 knockdown could also inhibit NLRP3, ASC, caspase-1, IL-1ß, and IL-18 in addition to suppressing cell apoptosis rate and promoting cell proliferation rate. Different expression pattern was found in cells transfected with H19 knockdown or CYP1B1 overexpression. Overexpression of CYP1B1 could abrogate the suppressive effect of H19 on pyroptosis of cardiomyocytes. H19 could inhibit activity of CYP1B1 promoters by regulating PBX3. CONCLUSION: H19 could inhibit CYP1B1 expression in a PBX3-dependent way and thus attenuate cell pyroptosis of cardiomyocytes.

Cytochrome P4501B1 in bone marrow is co-expressed with key markers of mesenchymal stem cells. BMS2 cell line models PAH disruption of bone marrow niche development functions.

Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous pollutants that are metabolized to carcinogenic dihydrodiol epoxides (PAHDE) by cytochrome P450 1B1 (CYP1B1). This metabolism occurs in bone marrow (BM) mesenchymal stem cells (MSC), which sustain hematopoietic stem and progenitor cells (HSPC). In BM, CYP1B1-mediated metabolism of 7, 12-dimethylbenz[a]anthracene (DMBA) suppresses HSPC colony formation within 6 h, whereas benzo(a)pyrene (BP) generates protective cytokines. MSC, enriched from adherent BM cells, yielded the bone marrow stromal, BMS2, cell line. These cells express elevated basal CYP1B1 that scarcely responds to Ah receptor (AhR) inducers. BMS2 cells exhibit extensive transcriptome overlap with leptin receptor positive mesenchymal stem cells (Lepr+ MSC) that control the hematopoietic niche. The overlap includes CYP1B1 and the expression of HSPC regulatory factors (Ebf3, Cxcl12, Kitl, Csf1 and Gas6). MSC are large, adherent fibroblasts that sequester small HSPC and macrophage in the BM niche (Graphic abstract). High basal CYP1B1 expression in BMS2 cells derives from interactions between the Ah-receptor enhancer and proximal promoter SP1 complexes, boosted by autocrine signaling. PAH effects on BMS2 cells model Lepr+MSC niche activity. CYP1B1 metabolizes DMBA to PAHDE, producing p53-mediated mRNA increases, long after the in vivo HSPC suppression. Faster, direct p53 effects, favored by stem cells, remain possible PAHDE targets. However, HSPC regulatory factors remained unresponsive. BP is less toxic in BMS2 cells, but, in BM, CYP1A1 metabolism stimulates macrophage cytokines (Il1b > Tnfa> Ifng) within 6 h. Although absent from BMS2 and Lepr+MSC, their receptors are highly expressed. The impact of this cytokine signaling in MSC remains to be determined.

Long-term exposure of 4-hydroxyestradiol induces the cancer cell characteristics via upregulating CYP1B1 in MCF-10A cells.

Life-long estrogen exposure is one of the major risk factors in the development and progression of breast cancer. However, little is known about the molecular mechanisms, by which chronic exposure to estrogen contributes to breast carcinogenesis. The aim of the present study was to investigate the effects of long-term exposure with 4-hydroxyestradiol (4-OHE2) on acquired cancer characteristics of human mammary epithelial MCF-10A cells. The possible regulators were further studied in chronic 4-OHE2-treated MCF-10A cells. We observed that MCF-10A cells long-term exposed to 4-OHE2 acquire the characteristics of cancer cells, such as enhanced cell growth, EMT properties, and increased migration and invasiveness. Moreover, the expression of CYP1B1 was significantly elevated in long-term 4-OHE2-treated MCF-10A cells. Block of CYP1B1 significantly reduced the cancer cell characteristics in long-term 4-OHE2-treated MCF-10A cells. Our results indicated that 4-OHE2 mediated enhanced cancer cell characteristics in mammary epithelial cells are an important key event for breast carcinogenesis process. CYP1B1 partially contributes to the 4-OHE2 induced cancer cell characteristics in MCF-10A cells. Targeting CYP1B1 might offer a new strategy for the treatment of estrogen-induced breast cancer.

Cyp1b1-deficient retinal astrocytes are more proliferative and migratory and are protected from oxidative stress and inflammation.

Astrocytes (ACs) are the most abundant cells in the central nervous system. Retinal ACs play an important role in maintaining the integrity of retinal neurovascular function, and their dysfunction contributes to the pathogenesis of various eye diseases including diabetic retinopathy. Cytochrome P450 1B1 (CYP1B1) expression in the neurovascular structures of the central nervous system including ACs has been reported. We previously showed that CYP1B1 expression is a key regulator of redox homeostasis in retinal vascular cells. Its deficiency in mice resulted in increased oxidative stress and attenuation of angiogenesis in vivo and proangiogenic activity of retinal vascular cells in vitro. Here, using retinal ACs prepared from wild-type (Cyp1b1+/+) and Cyp1b1-deficient (Cyp1b1-/-) mice, we determined the impact of Cyp1b1 expression on retinal AC function. We showed that Cyp1b1-/- retinal ACs were more proliferative and migratory. These cells also produced increased amounts of fibronectin and its receptors, αvß3- and α5ß1-integrin. These results were consistent with the increased adhesive properties of Cyp1b1-/- ACs and their lack of ability to form a network in Matrigel. This was reversed by reexpression of Cyp1b1 in Cyp1b1-/- ACs. Although no significant changes were observed in Akt/SRC/MAPK signaling pathways, production of inflammatory mediators bone morphogenetic protein-7 (BMP-7) and monocyte chemoattractant protein-1 (MCP-1) was decreased in Cyp1b1-/- ACs. Cyp1b1-/- ACs also showed increased levels of connexin 43 phosphorylation and cluster of differentiation 38 expression when challenged with H2O2. These results are consistent with increased proliferation and diminished oxidative stress in Cyp1b1-/- cells. Thus, Cyp1b1 expression in ACs plays an important role in retinal neurovascular homeostasis.

Cytochrome P450 1B1 Is Critical for Neointimal Growth in Wire-Injured Carotid Artery of Male Mice.

Background We have reported that cytochrome P450 1B1 ( CYP 1B1), expressed in cardiovascular tissues, contributes to angiotensin II -induced vascular smooth muscle cell ( VSMC ) migration and proliferation and development of hypertension in various experimental animal models via generation of reactive oxygen species. This study was conducted to determine the contribution of CYP 1B1 to platelet-derived growth factor-BB-induced VSMC migration and proliferation in vitro and to neointimal growth in vivo. Methods and Results VSMC s isolated from aortas of male Cyp1b1 +/+ and Cyp1b1 -/- mice were used for in vitro experiments. Moreover, carotid arteries of Cyp1b1 +/+ and Cyp1b1 -/- mice were injured with a metal wire to assess neointimal growth after 14 days. Platelet-derived growth factor- BB -induced migration and proliferation and H2O2 production were found to be attenuated in VSMC s from Cyp1b1 -/- mice and in VSMC s of Cyp1b1 +/+ mice treated with 4-hydroxy-2,2,6,6-tetramethylpiperidin-1-oxyl, a superoxide dismutase and catalase mimetic. In addition, wire injury resulted in neointimal growth, as indicated by increased intimal area, intima/media ratio, and percentage area of restenosis, as well as elastin disorganization and adventitial collagen deposition in carotid arteries of Cyp1b1 +/+ mice, which were minimized in Cyp1b1 -/- mice. Wire injury also increased infiltration of inflammatory and immune cells, as indicated by expression of CD 68+ macrophages and CD 3+ T cells, respectively, in the injured arteries of Cyp1b1 +/+ mice, but not Cyp1b1 -/- mice. Administration of 4-hydroxy-2,2,6,6-tetramethylpiperidin-1-oxyl attenuated neointimal growth in wire-injured carotid arteries of Cyp1b1 +/+ mice. Conclusions These data suggest that CYP 1B1-dependent oxidative stress contributes to the neointimal growth caused by wire injury of carotid arteries of male mice.

Effects of simvastatin on nuclear receptors, drug metabolizing enzymes and transporters expression in Human Umbilical Vein Endothelial Cells.

BACKGROUND: Vascular endothelial cells (EC) are constantly exposed to endo- and exogenous compounds, which may disturb EC function. One of the protecting mechanisms against chemicals consists of drug metabolizing enzymes and transporter proteins regulated by nuclear receptors and transcription factors. Therefore, the aim of the current study was to assess the regulation of nuclear receptors and their coordinated genes in Human Umbilical Vein Endothelial Cells (HUVEC). METHODS: HUVEC were exposed to TCDD (10nM), oltipraz (100µM) and simvastatin (1µM) for 24h. Gene expressions were evaluated using quantitative real-time PCR. The protein expression levels were determined by Western blotting. Enzymatic activity of CYP1A1/CYP1B1 was assessed by luciferin-labelled CYPs substrate. RESULTS: Our study confirmed that nuclear receptor AhR and nuclear factor Nrf2 are highly expressed in HUVECs. Treatment of HUVECs with TCDD (AhR inducer) resulted in a significant induction of AHR target genes CYP1A1, CYP1B1 and NQO1. Oltipraz (Nrf2 inducer) also markedly increased expression of NQO1 but did not affect Nrf2 mRNA nor protein levels. Under simvastatin stimulation PXR and NRF2 target transcripts were not altered, however AHR-regulated genes: CYP1A1, CYP1B1 and MDR1 were significantly induced. Western blot analysis confirmed CYP1B1 induction in TCDD-treated HUVECs, but not in the simvastatin group. Moreover, HUVEC exposure to TCDD resulted in induction of CYP1A1/CYP1B1 enzymatic activity. CONCLUSIONS: This study revealed functional expression of AhR and Nrf2 in HUVECs. Moreover, it was defined that simvastatin induced AhR and its related genes.

Malassezia pachydermatis up-regulates AhR related CYP1A1 gene and epidermal barrier markers in human keratinocytes.

Cytochrome P450 CYP1A1 and CYP1B1 enzymes are regulated by the aryl hydrocarbon receptor (AhR), a transcription factor activated by a variety of ligands among which Malassezia metabolites. In this study, we analyzed the modulation of CYP1A1, CYP1B1, and AhR in human keratinocytes infected with different strains of Malassezia pachydermatis, as well as the upregulation of some genes involved in the epidermal homeostasis. We demonstrated that all the strains induced AhR activation and its nuclear translocation in HaCaT cells infected for 24 h, compared to untreated cells. The expression of CYP1A1 and CYP1B1, prototypical markers of the AhR signaling pathway, were upregulated with the level of CYP1A1 mRNA approximately 100-fold greater than that for CYP1B1. Filaggrin, involucrin, and TGaseI, proteins involved in epidermal differentiation, were all modulated by Malassezia pachydermatis strains, with the strongest induction observed for filaggrin. By contrast, quinone oxidoreductase 1 (NQO1), which is part of the antioxidant defense system involved in detoxification, was not modulated in our experimental model. In conclusions, our findings suggest that Malassezia pachydermatis infection of human keratinocytes induces activation of the AhR, and increases the expression of its responsive genes and markers of epidermal differentiation, paving the way for occurrence/exacerbation of pathological skin conditions.

Effects of fuel components and combustion particle physicochemical properties on toxicological responses of lung cells.

The physicochemical properties of combustion particles that promote lung toxicity are not fully understood, hindered by the fact that combustion particles vary based on the fuel and combustion conditions. Real-world combustion-particle properties also continually change as new fuels are implemented, engines age, and engine technologies evolve. This work used laboratory-generated particles produced under controlled combustion conditions in an effort to understand the relationship between different particle properties and the activation of established toxicological outcomes in human lung cells (H441 and THP-1). Particles were generated from controlled combustion of two simple biofuel/diesel surrogates (methyl decanoate and dodecane/biofuel-blended diesel (BD), and butanol and dodecane/alcohol-blended diesel (AD)) and compared to a widely studied reference diesel (RD) particle (NIST SRM2975/RD). BD, AD, and RD particles exhibited differences in size, surface area, extractable chemical mass, and the content of individual polycyclic aromatic hydrocarbons (PAHs). Some of these differences were directly associated with different effects on biological responses. BD particles had the greatest surface area, amount of extractable material, and oxidizing potential. These particles and extracts induced cytochrome P450 1A1 and 1B1 enzyme mRNA in lung cells. AD particles and extracts had the greatest total PAH content and also caused CYP1A1 and 1B1 mRNA induction. The RD extract contained the highest relative concentration of 2-ring PAHs and stimulated the greatest level of interleukin-8 (IL-8) and tumor necrosis factor-alpha (TNFα) cytokine secretion. Finally, AD and RD were more potent activators of TRPA1 than BD, and while neither the TRPA1 antagonist HC-030031 nor the antioxidant N-acetylcysteine (NAC) affected CYP1A1 or 1B1 mRNA induction, both inhibitors reduced IL-8 secretion and mRNA induction. These results highlight that differences in fuel and combustion conditions affect the physicochemical properties of particles, and these differences, in turn, affect commonly studied biological/toxicological responses.

Cyp1b1 Regulates Ocular Fissure Closure Through a Retinoic Acid-Independent Pathway.

Purpose: Mutations in the CYP1B1 gene are the most commonly identified genetic causes of primary infantile-onset glaucoma. Despite this disease association, the role of CYP1B1 in eye development and its in vivo substrate remain unknown. In the present study, we used zebrafish to elucidate the mechanism by which cyp1b1 regulates eye development. Methods: Zebrafish eye and neural crest development were analyzed using live imaging of transgenic zebrafish embryos, in situ hybridization, immunostaining, TUNEL assay, and methylacrylate sections. Cyp1b1 and retinoic acid (RA) levels were genetically (morpholino oligonucleotide antisense and mRNA) and pharmacologically manipulated to examine gene function. Results: Using zebrafish, we observed that cyp1b1 was expressed in a specific spatiotemporal pattern in the ocular fissures of the developing zebrafish retina and regulated fissure patency. Decreased Cyp1b1 resulted in the premature breakdown of laminin in the ventral fissure and altered subsequent neural crest migration into the anterior segment. In contrast, cyp1b1 overexpression inhibited cell survival in the ventral ocular fissure and prevented fissure closure via an RA-independent pathway. Cyp1b1 overexpression also inhibited the ocular expression of vsx2, pax6a, and pax6b and increased the extraocular expression of shha. Importantly, embryos injected with human wild-type but not mutant CYP1B1 mRNA also showed colobomas, demonstrating the evolutionary and functional conservation of gene function between species. Conclusions: Cyp1b1 regulation of ocular fissure closure indirectly affects neural crest migration and development through an RA-independent pathway. These studies provide insight into the role of Cyp1b1 in eye development and further elucidate the pathogenesis of primary infantile-onset glaucoma.

Human T lymphocytes bioactivate heterocyclic aromatic amines by forming DNA adducts.

Heterocyclic aromatic amines (HAA) are formed in cooked meat, poultry and fish but also arise in tobacco smoke and exhaust gases. HAA are potential human carcinogens, which require metabolic activation to exert their genotoxicity. Human tissues can bioactivate HAA to produce reactive intermediates that bind to DNA. HAA DNA adduct formation occurs in human hepatocytes; however, the potential of HAA to form DNA adducts has not been investigated in human T lymphocytes. In this study, we investigated the ability of human T lymphocytes activated with PMA/Ionomycin or CD3/CD28 to express functional CYP1 activity and bioactivate three major HAA: 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx), and 2-amino-9H-pyrido[2,3-b]indole (AαC) to form DNA adducts. Adducts were measured by ultraperformance liquid chromatography-electrospray ionization/multistage scan mass spectrometry. The highest level of DNA adducts occurred for AαC (16 adducts per 109 nucleotides), followed by PhIP (9 adducts per 109 nucleotides). In contrast, DNA adducts formed from MeIQx and the structurally related aromatic amine 4-aminobiphenyl, a known human carcinogen, were below the limit of detection (< 3 adducts per 109 nucleotides). Moreover, we demonstrate that AαC is a potent inducer of CYP1A1 and CYP1B1 activity through a transcriptional mechanism involving the AhR pathway. Overall, our results highlight the capacity of activated human T lymphocytes to more efficiently bioactivate AαC to form DNA adducts than other prominent HAA or 4-ABP. Environ. Mol. Mutagen. 57:656-667, 2016. © 2016 Wiley Periodicals, Inc.

Role of aryl hydrocarbon receptor polymorphisms on TCDD-mediated CYP1B1 induction and IgM suppression by human B cells.

Previous studies have demonstrated that most of the intraspecies variation in sensitivity to the toxic effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), including suppression of antibody responses, in murine models is due to single nucleotide polymorphisms (SNPs) within the aryl hydrocarbon receptor (AhR) gene. The underlying reason for variation in sensitivity to TCDD-induced suppression of IgM responses among humans is not well understood, but is thought, in part, to be a result of different polymorphic forms of the AhR expressed by different individuals. In this study, the functional properties of six (P517S, R554K, V570I, V570I+P517S, R554K+V570I and P517S+R554K+V570I) human AhR variants were examined in the human B cell line, SKW 6.4. TCDD-induced Cyp1B1 and Cyp1A2 mRNA expression levels and Cyp1B1-regulated reporter gene activity, used for comparative purposes, were markedly lower in SKW cells containing the R554K SNP than in SKW-AHR(+) (control AhR) cells. Furthermore, all AhR variants were able to mediate TCDD-induced suppression of the IgM response; however, a combined P517S+R554K+V570I variant partially reduced sensitivity to TCDD-mediated suppression of IgM secretion. Collectively, our findings show that the R554K human AhR SNP alone altered sensitivity of human B cells to TCDD-mediated induction of Cyp1B1 and Cyp1A2. By contrast, attenuation of TCDD-induced IgM suppression required a combination of all three SNPs P517S, R554K, and V570I.

CYP1B1 Enhances Cell Proliferation and Metastasis through Induction of EMT and Activation of Wnt/ß-Catenin Signaling via Sp1 Upregulation.

Cytochrome P450 1B1 (CYP1B1) is a major E2 hydroxylase involved in the metabolism of potential carcinogens. CYP1B1 expression has been reported to be higher in tumors compared to normal tissues, especially in hormone-related cancers including breast, ovary, and prostate tumors. To explore the role of CYP1B1 in cancer progression, we investigated the action of CYP1B1 in cells with increased CYP1B1 via the inducer 7,12-dimethylbenz[α]anthracene (DMBA) or an overexpression vector, in addition to decreased CYP1B1 via the inhibitor tetramethoxystilbene (TMS) or siRNA knockdown. We observed that CYP1B1 promoted cell proliferation, migration, and invasion in MCF-7 and MCF-10A cells. To understand its molecular mechanism, we measured key oncogenic proteins including ß-catenin, c-Myc, ZEB2, and matrix metalloproteinases following CYP1B1 modulation. CYP1B1 induced epithelial-mesenchymal transition (EMT) and activated Wnt/ß-catenin signaling via upregulation of CTNNB1, ZEB2, SNAI1, and TWIST1. Sp1, a transcription factor involved in cell growth and metastasis, was positively regulated by CYP1B1, and suppression of Sp1 expression by siRNA or DNA binding activity using mithramycin A blocked oncogenic transformation by CYP1B1. Therefore, we suggest that Sp1 acts as a key mediator for CYP1B1 action. Treatment with 4-hydroxyestradiol (4-OHE2), a major metabolite generated by CYP1B1, showed similar effects as CYP1B1 overexpression, indicating that CYP1B1 activity mediated various oncogenic events in cells. In conclusion, our data suggests that CYP1B1 promotes cell proliferation and metastasis by inducing EMT and Wnt/ß-catenin signaling via Sp1 induction.

Cytochrome P450 1B1 Contributes to the Development of Atherosclerosis and Hypertension in Apolipoprotein E-Deficient Mice.

Cytochrome P450 (CYP) 1B1 contributes to vascular smooth muscle cell growth and hypertension in male mice. This study was conducted to determine the contribution of CYP1B1 to the development of atherosclerosis and hypertension and associated pathogenesis in 8-week-old male apolipoprotein E-deficient (ApoE(-/-)/Cyp1b1(+/+)), and ApoE- and CYP1B1-deficient (ApoE(-/-)/Cyp1b1(-/-)) mice fed a normal or atherogenic diet for 12 weeks. A separate group of ApoE(-/-)/Cyp1b1(+/+) mice on an atherogenic diet was injected every third day with the CYP1B1 inhibitor, 2,3',4,5'-tetramethoxystilbene (300 µg/kg), or its vehicle, dimethyl sulfoxide (30 µL, IP); systolic blood pressure was measured by the tail cuff method. After 12 weeks, mice were euthanized, blood collected for lipid analysis, and aortas harvested for measuring lesions and remodeling, and for infiltration of inflammatory cells by histological and immunohistochemical analysis, respectively, and for reactive oxygen species production. Blood pressure, areas of lipids and collagen deposition, elastin breaks, infiltration of macrophages and T lymphocytes, reactive oxygen species generation in the aorta, and plasma lipid levels were increased in ApoE(-/-)/Cyp1b1(+/+) mice on an atherogenic diet; these changes were minimized in mice given 2,3',4,5'-tetramethoxystilbene, and in ApoE(-/-)/Cyp1b1(-/-) mice on an atherogenic diet; absorption/production of lipids remained unaltered in these mice. These data suggest that aortic lesions, hypertension, and associated pathogenesis in ApoE(-/-)/Cyp1b1(+/+) mice on an atherogenic diet are most likely dependent on CYP1B1-generated oxidative stress and increased plasma lipid levels independent of blood pressure and absorption of lipids. CYP1B1 could serve as a novel target for developing drugs to treat atherosclerosis and hypertension caused by hypercholesterolemia.

Determination of Heavy Metal Concentrations in Normal and Pathological Human Endometrial Biopsies and In Vitro Regulation of Gene Expression by Metals in the Ishikawa and Hec-1b Endometrial Cell Line.

It is well known that several metals, such as lead, mercury, cadmium, and vanadium, can mimic the effects of estrogens (metallo-estrogens). Nevertheless, there are only a few studies that have assessed the effects of toxic metals on the female genital tract and, in particular, endometrial tissue. In this context, we measured the concentrations of several trace elements in human endometrial tissue samples from individuals with hyperplasia or adenocarcinoma and in normal tissues. Hyperplasic endometrial tissue has a 4-fold higher concentration of mercury than normal tissue. Mercury can affect both the AhR and ROS signaling pathways. Thus, we investigated the possible toxic effects of mercury by in vitro studies. We found that mercury increases oxidative stress (increased HO1 and NQO1 mRNA levels) and alters the cytoskeleton in the human endometrial Ishikawa cell line and to a lesser extent, in the "less-differentiated" human endometrial Hec-1b cells. The results might help to explain a potential link between this metal and the occurrence of endometrial hyperplasia.

Differences in 4-hydroxyestradiol levels in leukocytes are related to CYP1A1(∗)2C, CYP1B1(∗)3 and COMT Val158Met allelic variants.

Exposure to estrogen and its metabolites, including catechol estrogens (CEs) and catechol estrogen quinones (CE-Qs) is closely related to breast cancer. Polymorphisms of the genes involved in the catechol estrogens metabolism pathway (CEMP) have been shown to affect the production of CEs and CE-Qs. In this study, we measured the induction of CYP1A1, CYP1B1, COMT, and GSTP1 by 17ß-estradiol (17ß-E2) in leukocytes with CYP1A1(∗)2C, CYP1B1(∗)3, COMT Val158Met and GSTP1 Ile105Val polymorphisms by semi quantitative RT-PCR and compared the values to those of leukocytes with wild type alleles; we also compared the differences in formation of 4- hydroxyestradiol (4-OHE2) and DNA-adducts. The data show that in the leukocytes with mutant alleles treatment with 17ß-E2 up-regulates CYP1A1 and CYP1B1 and down-regulates COMT mRNA levels, resulting in major increments in 4-OHE2 levels compared to leukocytes with wild-type alleles. Therefore, we propose induction levels of gene expression and intracellular 4-OHE2 concentrations associated with allelic variants in response to exposure of 17ß-E2 as a noninvasive biomarker that can help determine the risk of developing non-hereditary breast cancer in women.

Stability of the aryl hydrocarbon receptor and its regulated genes in the low activity variant of Hepa-1 cell line.

We examined the expression kinetics of some of the aryl hydrocarbon receptor (AhR)-regulated genes in LA1 variant cells compared to wild type (WT) Hepa-1 mouse hepatoma cell lines, and we investigated the stability of AhR protein as a key step in the function of this receptor. Treatment of both cell types with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) resulted in increased CYP1A1 and CYP1B1 mRNA with a subsequent down regulation of AhR. We show here that co-treatment with transcription inhibitor actinomycin D (ActD) has reversed the TCDD-induced depletion of AhR protein in WT. However, the proteolytic degradation of AhR in absence of TCDD was significantly higher in LA1 cells than in WT, and ActD treatment reduced this loss. Induction of CYP1A1 and CYP1B1 mRNA by TCDD in WT cells each exhibited bursts of activity in the initial hour which were about 3-fold greater than in LAI cells. The induced mRNA levels in LA1 exhibited a slow and sustained increase approximating the WT levels by 20h. The induction of two other AhR-regulated genes also showed comparable turnover differences between the two types of cell. Thus, altered regulation of the AhR responsive genes in LA1 may result from a difference in AhR stability.

Analysis of the AHR gene proximal promoter GGGGC-repeat polymorphism in lung, breast, and colon cancer.

The aryl hydrocarbon receptor (AhR) regulates expression of numerous genes, including those of the CYP1 gene family. With the goal of determining factors that control AHR gene expression, our studies are focused on the role of the short tandem repeat polymorphism, (GGGGC)n, located in the proximal promoter of the human AHR gene. When luciferase constructs containing varying GGGGC repeats were transfected into cancer cell lines derived from the lung, colon, and breast, the number of GGGGC repeats affected AHR promoter activity. The number of GGGGC repeats was determined in DNA from 327 humans and from 38 samples representing 5 species of non-human primates. In chimpanzees and 3 species of macaques, only (GGGGC)2 alleles were observed; however, in western gorilla, (GGGGC)n alleles with n=2, 4, 5, 6, 7, and 8 were identified. In all human populations examined, the frequency of (GGGGC)n was n=4>5≫2, 6. When frequencies of the (GGGGC)n alleles in DNA from patients with lung, colon, or breast cancer were evaluated, the occurrence of (GGGGC)2 was found to be 8-fold more frequent among lung cancer patients in comparison with its incidence in the general population, as represented by New York State neonates. Analysis of matched tumor and non-tumor DNA samples from the same individuals provided no evidence of microsatellite instability. These studies indicate that the (GGGGC)n short tandem repeats are inherited, and that the (GGGGC)2 allele in the AHR proximal promoter region should be further investigated with regard to its potential association with lung cancer susceptibility.

Hyperoxia-induced changes in estradiol metabolism in postnatal airway smooth muscle.

Supplemental oxygen, used to treat hypoxia in preterm and term neonates, increases the risk of neonatal lung diseases, such as bronchopulmonary dysplasia (BPD) and asthma. There is a known sex predilection for BPD, but the underlying mechanisms are not clear. We tested the hypothesis that altered, local estradiol following hyperoxia contributes to pathophysiological changes observed in immature lung. In human fetal airway smooth muscle (fASM) cells exposed to normoxia or hyperoxia, we measured the expression of proteins involved in estrogen metabolism and cell proliferation responses to estradiol. In fASM cells, CYP1a1 expression was increased by hyperoxia, whereas hyperoxia-induced enhancement of cell proliferation was blunted by estradiol. Pharmacological studies indicated that these effects were attributable to upregulation of CYP1a1 and subsequent increased metabolism of estradiol to a downstream intermediate 2-methoxyestradiol. Microarray analysis of mouse lung exposed to 14 days of hyperoxia showed the most significant alteration in CYP1a1 expression, with minimal changes in expression of five other genes related to estrogen receptors, synthesis, and metabolism. Our novel results on estradiol metabolism in fetal and early postnatal lung in the context of hyperoxia indicate CYP1a1 as a potential mechanism for the protective effect of estradiol in hyperoxia-exposed immature lung, which may help explain the sex difference in neonatal lung diseases.

CYP1B1 enhances the resistance of epithelial ovarian cancer cells to paclitaxel in vivo and in vitro.

Ovarian cancer (OC) is the most frequent cause of mortality among gynecological malignancies, with a 5-year survival rate of approximately 30%. The standard regimen for OC therapy includes a platinum agent combined with a taxane, to which the patients frequently acquire resistance. Resistance arises from the oxidation of anticancer drugs by CYP1B1, a cytochrome P450 enzyme overexpressed in malignant OC. The aim of the present study was to determine the role of CYP1B1 expression in the drug resistance of OC to the taxane, paclitaxel (PTX). Immunohistochemical staining was used to assess CYP1B1 expression in a panel of ovarian samples (53 primary cancer samples, 14 samples of metastastic cancer, 30 benign tumor samples and 19 normal tissue samples). Semi-quantitative RT-PCR was also performed to determine CYP1B1 expression in several OC cell lines. Finally, we used proliferation and toxicity assays, as well as a mouse xenograft model using nude mice to determine whether α-naphthoflavone (ANF), a CYP1B1 specific inhibitor, reduces resistance to PTX. CYP1B1 was overexpressed in the samples from primary and metastatic loci of epithelial ovarian cancers. In some cell lines, PTX induced CYP1B1 expression, which resulted in drug resistance. Exposure to ANF reduced drug resistance and enhanced the sensitivity of OC cells to PTX in vitro and in vivo. The expression profile of CYP1B1 suggests that it has the potential to be a useful diagnostic marker and prognostic factor for malignant OC. The inhibition of CYP1B1 expression by specific agents may provide a novel therapeutic strategy for the treatment of patients resistant to PTX and may improve the prognosis of these patients.