The 4-hydroxy metabolite of 178-estradiol (E2) has been implicated in the carcinogenicity of this hormone. Previous studies showed that aryl hydrocarbonreceptor agonists induced a cytochrome P450 that catalyzed the 4-hydroxylation of E2. This activity was associated with human P450 lBi. To determine the relationship of the human P450 lBl 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 Km values of 0.71 and 0.78 ,uM and turnover numbers of 1.39 and 0.27 nmol product min'l 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 lBl and P450 lAl mRNA levels, and caused increased rates of 2-, 4-, 6c-, and 15a-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 Km P450 lBl-component of E2 metabolism. These studies demonstrate that human P450 lB1 is a catalytically efficient E2 4-hydroxylase that is likely to participate in endocrine regulation and the toxicity of estrogens.The importance of estrogens in the etiology of breast and uterine cancer is widely recognized (1-3). The carcinogenicity of estrogens has been primarily attributed to their action as agonists of the estrogen receptor, through which concerted gene regulation controls cellular growth and differentiation in estrogen responsive tissues. Increasing evidence of another mechanism of carcinogenicity has focused attention on the catechol estrogen metabolites, which are less potent estrogens than 17f3-estradiol (E2). The 2-and 4-hydroxylated metabolites of both E2 and estrone (E1) can directly or indirectly damage DNA, proteins, and lipids through the generation of reactive free radicals by the reductive-oxidative cycling of these catechol estrogens between their semiquinone and quinone forms (4-6).4-Hydroxylated metabolites represent only a small percentage of the total urinary catechol-estrogen content, and 4-hydroxylation was previously thought to be only a minor metabolic route (7). However, tissue-specific 4-hydroxylation of E2 may be significant in the metabolic control of estrogen homeostasis. In human (8) and mouse uteri (9), rat pituitary (10), and hamster kidney (11) the rate of E2 4-hydroxylation approaches or exceeds that of 2-hydroxylation. Interestingly, these organs are targets of estrogen-induced tumorigenesis (2, 12-14), and higher E2 4-hydroxylase activity has been measured in tumors of the human breast (15, 16) and uterus (8), each compared with normal tissue. Furthermore, in the male hamster kidney, the carcinogenic and DNA-damaging activity of 4-hydroxyestradiol (4-OHE2), and lack of activity of 2-hydroxyestradiol (2-OHE2), (17)(18)(19), implicate the 4-hydroxylated metabolites in estrogen-induced carcinogenesis. Pertinent to elucidating the contribution of 4...

Human cytochromes P450 1A1 (CYP1A1) and P450 1B1 (CYP1B1) catalyze the metabolic activation of a number of procarcinogens and the hydroxylation of 17beta-estradiol (E2) at the C-2 and C-4 positions, respectively. The aromatic hydrocarbon receptor (AhR) agonist 2,3,7,8-tetrachlorodibenzo-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-10A) 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) alpha mRNA, CYP1A1 mRNA and activity were induced by TCDD, and enhanced E2 metabolism in TCDD-treated cells was predominantly E2 2-hydroxylation. In MCF-10A, MCF-7, T-47D, ZR-75-1 and BT-20 cells, which expressed varying levels of ER alpha 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 ER alpha 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.

Hypoxia-inducible factor 1 (HIF-1) is a transcription factor that mediates cellular and systemic homeostatic responses to reduced O2 availability in mammals, including angiogenesis, erythropoiesis, and glycolysis. HIF-1 activity is controlled by the O 2-regulated expression of the HIF-1␣ subunit. Under nonhypoxic conditions, HIF-1␣ protein is subject to ubiquitination and proteasomal degradation. Here we report that missense mutations and͞or deletions involving several different regions of HIF-1␣ result in constitutive expression and transcriptional activity in nonhypoxic cells. We demonstrate that hypoxia results in decreased ubiquitination of HIF-1␣ and that missense mutations increase HIF-1␣ expression under nonhypoxic conditions by blocking ubiquitination.

Chloracne is commonly observed in humans exposed to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD); yet, the mechanism of toxicity is not well understood. Using normal human epidermal keratinocytes, we investigated the mechanism of TCDD-mediated enhancement of epidermal differentiation by integrating functional genomic, metabolomic, and biochemical analyses. TCDD increased the expression of 40% of the genes of the epidermal differentiation complex found on chromosome 1q21 and 75% of the genes required for de novo ceramide biosynthesis. Lipid analysis demonstrated that eight of the nine classes of ceramides were increased by TCDD, altering the ratio of ceramides to free fatty acids. TCDD decreased the expression of the glucose transporter, SLC2A1, and most of the glycolytic transcripts, followed by decreases in glycolytic intermediates, including pyruvate. NADH and Krebs cycle intermediates were decreased, whereas NAD(+) was increased. Mitochondrial glutathione (GSH) reductase activity and the GSH/glutathione disulfide ratio were decreased by TCDD, ultimately leading to mitochondrial dysfunction, characterized by decreased inner mitochondrial membrane potential and ATP production, and increased production of the reactive oxygen species (ROS), hydrogen peroxide. Aryl hydrocarbon receptor (AHR) antagonists blocked the response of many transcripts to TCDD, and the endpoints of decreased ATP production and differentiation, suggesting regulation by the AHR. Cotreatment of cells with chemical antioxidants or the enzyme catalase blocked the TCDD-mediated acceleration of keratinocyte cornified envelope formation, an endpoint of terminal differentiation. Thus, TCDD-mediated ROS production is a critical step in the mechanism of this chemical to accelerate keratinocyte differentiation.