Differential expression of CYP1A1 and CYP1B1 in human breast epithelial cells and breast tumor cells

Human cytochromes P450 1A1 (CYP1A1) and P450 1B1 (CYP1B1) catalyze the metabolic activation of a number of procarcinogens and the hydroxylation of 17β-estradiol (E2) at the C-2 and C-4 positions, respectively. The aromatic hydrocarbon receptor (AhR) agonist 2,3,7,8-tetrachlorodi-benzo-p-dioxin (TCDD) has a marked effect on estrogen metabolism in MCF-7 breast-tumor cells by induction of these two enzymes. To investigate whether induction of CYP1A1 and CYP1B1 by AhR agonists and the associated increase in E2 metabolism are common to all breast epithelial cells and breast-tumor cells, we determined the effects of TCDD on E2 metabolism, and CYP1A1 and CYP1B1 mRNA levels in a series of non-tumor-derived breast epithelial (184A1 and MCF-1OA) and breast-tumor (MCF-7, T-47D, ZR-75-1, BT-20, MDA-MB-157, MDA-MB-231 and MDA-MB-436) cell lines. In 184A1 cells, which did not express detectable estrogen receptor (ER) α mRNA, CYP1A1 mRNA and activity were induced by TCDD, and enhanced E2 metabolism in TCDD-treated cells was predominantly E2 2-hydroxylation. In MCF-1OA, MCF-7, T-47D, ZR-75-1 and BT-20 cells, which expressed varying levels of ERα mRNA, both CYP1A1 and CYP1B1 mRNA levels and rates of both E2 2- and 4-hydroxylation were highly elevated following exposure to TCDD. In MDA-MB-157, MDA-MB-231 and MDA-MB-436 cells, which did not express detectable ERα mRNA and generally displayed fibroblastic or mesenchymal rather than epithelial morphology, CYP1B1 induction was favored, and the rate of E2 4-hydroxylation exceeded that of 2-hydroxylation in TCDD-treated cells. These results show that breast epithelial cells and tumor cells vary widely with regard to AhR-mediated CYP1A1 and CYP1B1 induction, suggesting that factors in addition to the AhR regulate CYP1A1 and CYP1B1 gene expression. In these cell lines, significant CYP1A1 inducibility was restricted to cultures displaying epithelial morphology, whereas CYP1B1 inducibility was observed in cells of both epithelial and mesenchymal morphology

Induction of cytochrome P450 1B1 and catechol estrogen metabolismin ACHN human renal adrenocarcinoma cells

The catechol estrogen metabolites of 17β-estradiol (E2), 2-hydroxyestradiol (OHE2) and 4-OHE2, differ in hormonal properties and carcinogenic potential. In Syrian hamster kidney, 4-OHE2 induces clear-cell carcinoma whereas 2-OHE2 does not, and an E2 4-hydroxylase appears to be involved in E2-induced carcinogenesis in these animals. specific E2 4-hydroxylase activity has been observed in extrahepatic tissues from several species. In humans, cytochrome P450 1B1 (CYP1B1) appears to be an extrahepatic E2 4-hydroxylase under the regulatory control of the aromatic hydrocarbon receptor (AhR). As an initial approach to investigating CYP1B1 expression and E2 4-hydroxylase activity in human kidney, we used the ACHN cell line, derived from a human renal adenocarcinoma. In untreated ACHN cells, a very low level of CYP1B1 mRNA expression was observed and CYP1B1 protein could not be detected; however, in ACHN cells exposed to the high-affinity AhR ligand, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), CYP1B1 mRNA levels were elevated 28-fold, and the CYP1B1 protein was detected by immunoblot analysis. Exposure of ACHN cells to TCDD resulted in minimal induction of the CYP1A1 mRNA, and the CYP1A1 protein was not detectable prior to or after exposure to TCDD. E2 hydroxylase activity could not detected with microsomes from untreated ACHN cells, although activities at C-4 and, to a lesser extent, at C-2 of E2 were observed with microsomes from TCDD-treated ACHN cells. In experiments with intact ACHN cells, elevated rates of formation of 4-methoxyestradiol (MeOE2) and 2-MeOE2 were observed in response to treatment with TCDD. The EC50 for induction of the CYP1B1 mRNA was 1.5 nM TCDD; EC50s for the stimulation of 2- and 4-MeOE2 formation were 0.68 and 1.1 nM TCDD. These results indicate that the ACHN cell line may be a useful in vitro model system to study the regulation of CYP1B1 expression and the cytotoxic effects associated with E2 4-hydroxylation

17β-Estradiol hydroxylation catalyzed by human cytochrome P450 1B1

The 4-hydroxy metabolite of 17β-estradiol (E2) has been implicated in the carcinogenicity of this hormone. Previous studies showed that aryl hydrocarbon-receptor agonists induced a cytochrome P450 that catalyzed the 4- hydroxylation of E2. This activity was associated with human P450 1B1. To determine the relationship of the human P450 1B1 gene product and E2 4- hydroxylation, the protein was expressed in Saccharomyces cerevisiae. Microsomes from the transformed yeast catalyzed the 4- and 2-hydroxylation of E2 with K(m) values of 0.71 and 0.78 μM and turnover numbers of 1.39 and 0.27 nmol product min-1 · nmol P450-1, respectively. Treatment of MCF-7 human breast cancer cells with the aryl hydrocarbon-receptor ligand indolo[3,2-b]carbazole resulted in a concentration-dependent increase in P450 1B1 and P450 1A1 mRNA levels, and caused increased rates of 2-, 4-, 6α-, and 15α-hydroxylation of E2. At an E2 concentration of 10 nM, the increased rates of 2- and 4-hydroxylation were approximately equal, emphasizing the significance of the low K(m) P450 1B1-component of E2 metabolism. These studies demonstrate that human P450 1B1 a catalytically efficient E2 4- hydroxylase that is likely to participate in endocrine regulation and the toxicity of estrogens

Inductive and inhibitory effects of non-ortho-substituted polychlorinated biphenyls on estrogen metabolism and human cytochromes P450 1A1 and 1B1

The effects of a series of non-ortho-substituted polychlorinated biphenyls (PCBs) on human cytochrome P450 1A1 (CYP1A1), a 17β-estradiol (E2) 2-hydroxylase, and P450 1B1 (CYP1B1), an E2 4-hydroxylase, were investigated in HepG2 and MCF-7 cells. Elevated rates of 2- and 4-methoxyestradiol (2- and 4-MeOE2) formation in PCB-treated cultures were measured as activities of CYP1A1 and CYP1B1, respectively. Of the congeners investigated, 3,4,4\u27,5-tetrachlorobiphenyl (PCB 81), 3,3\u27,4,4\u27,5-pentachlorobiphenyl (PCB 126), and 3,4\u27,5-trichlorobiphenyl (PCB 39) caused marked stimulation of E2 metabolism in both cell lines. Northern blot analyses confirmed that exposure of MCF-7 cells to PCBs 81, 126, and 39 caused highly elevated levels of the CYP1A1 and CYP1B1 mRNAs. Exposure of MCF-7 cells to 3,3\u27,4,4\u27,5,5\u27-hexachlorobiphenyl (PCB 169) resulted in elevated levels of the CYP1A1 and CYP1B1 mRNAs, but did not cause elevated rates of E2 metabolism; rather, 4-MeOE2 production was depressed to below control levels in PCB 169-treated cultures. PCB 169 also inhibited the 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-induced 4-MeOE2 and, to a lesser extent, 2-MeOE2 production in MCF-7 cells, as did PCB 126 and several other congeners. In microsomal assays, inhibition of cDNA-expressed human CYP1B1 by PCBs 169 and 126 was demonstrated. These studies with one subgroup of PCBs, the non-ortho-substituted congeners, underscore the complexity and diversity of effects of PCBs, as individual congeners were found both to induce expression and to inhibit activity of human CYP1B1 and CYP1A1. Copyright (C) 1999 Elsevier Science Inc

Research governance: implications for health library and information professionals

The Research Governance Framework for Health and Social Care published by the Department of Health in 2001 provides a model of best practice and a framework for research in the health and social care sector. This article reviews the Department of Health Research Governance Framework, discusses the implications of research governance for library and information professionals undertaking research in the health- and social-care sector and recommends strategies for best practice within the information profession relating to research governance. The scope of the Framework document that covers both clinical and non-clinical research is outlined. Any research involving, amongst other issues, patients, NHS staff and use or access to NHS premises may require ethics committee approval. Particular reference is made to the roles, responsibilities and professional conduct and the systems needed to support effective research practice. Issues such as these combine to encourage the development of a quality research culture which supports best practice. Questions arise regarding the training and experience of researchers, and access to the necessary information and support. The use of the Framework to guide research practice complements the quality issues within the evidence-based practice movement and supports the ongoing development of a quality research culture. Recommendations are given in relation to the document's five domains of ethics, science, information, health and safety and finance and intellectual property. Practical recommendations are offered for incorporating research governance into research practice in ways which conform to the Framework's standards and which are particularly relevant for research practitioners in information science. Concluding comments support the use of the Research Governance Framework as a model for best practice