Metabolism of equilenin in MCF-7 and MDA-MB-231 human breast cancer cells.

Sulfate conjugates of the B-ring unsaturated estrogens, equilin, equilenin, and 8-dehydroestrone, and their 17alpha- and 17beta-dihydro analogues, constitute about 54% of Premarin (Wyeth-Ayerst), the most commonly prescribed estrogen formulation in estrogen replacement therapy. Despite the wide clinical use of Premarin, there have been very few studies on the metabolism of the B-ring unsaturated estrogens in humans and there is no information regarding the fate of these compounds in breast tissue or tumors. In this study, we investigated the metabolism of equilenin in two lines of human breast-cancer cells, MCF-7 and MDA-MB-231. MCF-7 cells respond to treatment with Ah-receptor agonists with induction of cytochromes P450 1A1 and 1B1, whereas in MDA-MB-231 cells P450 1B1 is predominantly induced. Metabolites of equilenin were identified and quantified by GC/MS utilizing a series of synthetic metabolite standards and deuterium-labeled analogues as internal standards. In the two cell lines, the same pathways of equilenin metabolism were observed. Equilenin was reduced at C-17 to the 17beta-dihydro form, with minimal production of the 17alpha-dihydro isomer. Both equilenin and 17beta-dihydroequilenin were hydroxylated at the C-4 position, and the resultant catechol metabolites were methylated to form 4-methoxyequilenin and 4-methoxy-17beta-dihydroequilenin. Rates of equilenin metabolism were markedly elevated in cultures exposed to the Ah-receptor agonists, 2,3,7,8-tetrachlorodibenzo-p-dioxin and 3,4,4',5-tetrachlorobiphenyl, implicating the activities of P450s 1A1 and 1B1 in the metabolism. The 2-hydroxylation pathways of equilenin and 17beta-dihydroequilenin metabolism were not observed. In microsomal reactions with cDNA-expressed human enzymes, both P450s 1A1 and 1B1 catalyzed the 4-hydroxylation of 17beta-dihydroequilenin, whereas with 17beta-estradiol as substrate P450 1A1 catalyzes predominantly 2-hydroxylation and P450 1B1 predominantly 4-hydroxylation. Since P450 1B1 is constitutively expressed and both P450s 1A1 and 1B1 are inducible in many extrahepatic tissues including the mammary epithelium, these results indicate the potential for 4-hydroxylation of equilenin and 17beta-dihydroequilenin in extrahepatic, estrogen-responsive tissues.

SULT1A1 catalyzes 2-methoxyestradiol sulfonation in MCF-7 breast cancer cells.

In a previous study of nine human breast-derived cell lines, rates of metabolism of 17beta-estradiol (E(2)) were greatly enhanced when cultures were exposed to the aromatic hydrocarbon receptor agonist, 2,3,7,8-tetrachlorodibenzo-p-dioxin. Elevated rates of E(2) hydroxylation at the C-2, -4, -6alpha and -15alpha positions were observed concomitant with the induction of cytochromes P450 1A1 and 1B1. In each cell line, 2- and 4-hydroxyestradiol (2- and 4-OHE(2)) were converted to 2- and 4-methoxyestradiol (2- and 4-MeOE(2)) by the action of catechol O:-methyltransferase. In this study, conjugation of these estrogen metabolites was investigated. A comparison of the levels of metabolites determined with and without prior treatment of the media with a crude beta-glucuronidase/sulfatase preparation showed that most of the 2-MeOE(2) present was in conjugated form, whereas 4-MeOE(2), 6alpha-OHE(2) and 15alpha-OHE(2) were minimally conjugated. Inhibitor studies suggested that it was the sulfatase activity of the preparation that hydrolyzed the 2-MeOE(2) conjugates in MCF-7 cell media; the presence of 2-MeOE(2)-3-sulfate in MCF-7 culture media was confirmed by electrospray ion-trap mass spectrometry. To identify the enzyme catalyzing this conjugation, the expression of mRNAs encoding five sulfotransferases (SULT1A1, SULT1A2, SULT1A3, SULT1E1 and SULT2A1) was evaluated in the nine cell lines by use of the reverse transcription-polymerase chain reaction. Only expression of SULT1A1 mRNA correlated with the observed conjugation of nanomolar levels of 2-MeOE(2) in these cell lines. Cloning and sequencing of SULT1A1 cDNA from MCF-7 cells revealed that mRNAs encoding two previously identified allelic variants, SULT1A1*1 ((213)Arg) and SULT1A1*2 ((213)His), were expressed in these cells. Heterologous cDNA-directed expression of either variant in MDA-MB-231 cells, which do not normally express SULT1A1, conferred 2-MeOE(2) sulfonation activity. The SULT1A1 allelic variants were also expressed in SF:9 insect cells, from which post-microsomal supernatants were used to determine K:(m) values of 0.90 +/- 0.12 and 0.81 +/- 0.06 microM for SULT1A1*1 and SULT1A1*2, respectively, with 2-MeOE(2) as substrate. These results show that SULT1A1 is an efficient and selective catalyst of 2-MeOE(2) sulfonation and, as such, may be important in modulating the anticarcinogenic effects of 2-MeOE(2) that have been described recently.

Growth inhibition of MCF-7 cells by estrogen is dependent upon a serum factor.

MCF-7 cell growth is normally dependent upon estrogen, but if cells are maintained in serum-free medium estrogen inhibits cell growth with an IC50 of about 1 nM. Cells adapted to serum-free medium have approximately the same levels of estrogen receptor mRNA as control cells that are stimulated by estrogen. We have identified two serum components, one estrogen dependent and one not, that appear to be responsible for the inhibition of growth by estrogen. Our observations are consistent with the presence of a plasma membrane receptor which negatively regulates MCF-7 cell growth, and that can be inhibited by a serum protein that binds estrogen.

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 17beta-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',5-tetrachlorobiphenyl (PCB 81), 3,3',4,4',5-pentachlorobiphenyl (PCB 126), and 3,4',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',4,4',5,5'-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.

Homologous Streptomycin Resistance Gene Present among Diverse Gram-Negative Bacteria in New York State Apple Orchards.

The streptomycin resistance gene of Pseudomonas syringae pv. papulans Psp36 was cloned into Escherichia coli and used to develop a 500-bp DNA probe that is specific for streptomycin resistance in P. syringae pv. papulans. The probe is a portion of a 1-kb region shared by three different DNA clones of the resistance gene. In Southern hybridizations, the probe hybridized only with DNA isolated from streptomycin-resistant strains of P. syringae pv. papulans and not with the DNA of streptomycin-sensitive strains. Transposon insertions within the region of DNA shared by the three clones resulted in loss of resistance to streptomycin. Colony hybridization of bacteria isolated from apple leaves and orchard soil indicated that 39% of 398 streptomycin-resistant bacteria contained DNA that hybridized to the probe. These included all strains of P. syringae pv. papulans and some other fluorescent pseudomonads and nonfluorescent gram-negative bacteria, but none of the gram-positive bacteria. The same-size restriction fragments hybridized to the probe in P. syringae pv. papulans. Restriction fragment length polymorphism of this region was occasionally observed in strains of other taxonomic groups of bacteria. In bacteria other than P. syringae pv. papulans, the streptomycin resistance probe hybridized to different-sized plasmids and no relationship between plasmid size and taxonomic group or between plasmid size and orchard type, soil association, or leaf association could be detected.

Use of ti plasmid DNA probes for determining tumorigenicity of agrobacterium strains.

Probes consisting of T-DNA genes from the Ti plasmid of Agrobacterium tumefaciens were used for determining tumorigenicity of strains. Two P-labeled probes hybridized with 28 of 28 tumorigenic strains of the pathogen but not with 20 of 22 nontumorigenic strains. One probe, pTHE17, consists of all but the far left portion of the T-DNA of strain C58. Probe SmaI7 consists of SmaI fragment 7 of pTiC58, including onc genes 1, 4, and 6a and most of 2. Another probe, pAL4044, consisting of the vir region of strain Ach-5, hybridized with several nontumorigenic as well as tumorigenic strains. Colony hybridizations were done with 28 tumorigenic and 22 nontumorigenic Agrobacterium strains. About 10 CFU of the different tumorigenic strains were detectable with this method. Southern analyses confirmed the presence or absence of Ti plasmids in strains for which tumorigenicity was questioned. Colony hybridization with the T-DNA probes provides a rapid and sensitive means for determining the tumorigenic nature of Agrobacterium strains.

Comparison of gradient-recalled-echo and T2-weighted spin-echo pulse sequences in intramedullary spinal lesions.

Nineteen consecutive patients with spinal intramedullary lesions were studied on a 1.5-T system to compare the quality of T2-weighted spin-echo and gradient-recalled-echo (GRE) pulse sequences. Direct comparisons were made in the sagittal and/or axial planes. Twenty-four studies were performed in the 19 patients. The gradient echoes were usually performed at 300/14 (TR/TE) with a flip angle of 10 degrees. Although no significant diagnostic differences were noted in the sagittal plane, there was a distinct anatomic advantage for GRE imaging over spin-echo imaging in the axial plane. This is believed to be the result of CSF time-of-flight effects in the slice-select direction, which are not compensated for by flow-compensating gradients on the spin-echo images, but which are insignificant in the GRE sequence used in this study. Pathology was seen equally well or better on GRE in 79% (19/24) of the sequences. In the other five cases, the spin-echo image showed a brighter intramedullary signal than that seen on GRE, although GRE showed the lesion in all cases. Our results indicate that properly optimized GRE imaging on a high-field-strength system can replace spin-echo imaging in the spine when intramedullary disease is suspected and that the benefits of GRE are most striking in the axial plane.

MR imaging of intracranial carotid occlusion.

The MR scans of seven patients with intracranial carotid occlusion (five proved, two presumed) were reviewed to evaluate the MR signal characteristics seen in this disorder. Five patients had clinical signs of cerebral infarction. Of the remaining two patients, one was asymptomatic and the other had a long-standing occlusion and headaches. We correlated the MR findings with cerebral angiography in five patients and with CT scans in six patients. All occluded vessels demonstrated MR signal predominantly isointense to brain on proton-density- T1- and T2-weighted images. Since there is an absence of flow, the MR signal is based on the intrinsic properties of the arterial thrombus and possibly on the chronicity of the occlusion. The pathogenesis and histopathology of intravascular thrombus differ significantly from extravascular hematoma, which accounts for the differences in their MR signal characteristics. The demonstration of occluded intracranial vessels may solidify the diagnosis of stroke in cases in which clinical and/or CT findings are equivocal. In patients presenting with infarction, an occluded carotid artery by MR may obviate the need for angiography; however, the demonstration of a patent carotid in conjunction with infarction suggests the possibility of an embolus, which may require angiography. We believe that MR is a valuable adjunct to CT in evaluating patients with cerebrovascular infarction.

Spinal infection: evaluation with MR imaging and intraoperative US.

Magnetic resonance (MR) images of the spine and/or intraoperative spinal ultrasound (US) in 24 patients with spinal infections were reviewed and correlated with clinical and pathologic data to determine their diagnostic value. In disk space infection with osteomyelitis and in retrospinal abscess, MR images showed characteristic findings, whereas in myelitis, MR images demonstrated nonspecific abnormalities. The appearance on MR images of epidural abscesses ranged from clearly identifiable extradural masses with high-intensity signal on spin-echo T2-weighted images to extensive inhomogeneous collections of mixed signal intensities, difficult to distinguish from adjacent meningitis. Myelography with high-resolution computed tomography (CT) and intraoperative spinal US was superior to MR imaging in demonstrating epidural abscesses when there was concomitant meningitis. With intraoperative spinal US, epidural abscesses could be located and their decompression monitored. MR imaging is recommended as the initial screening procedure in spinal infection; in those few patients with nondiagnostic MR images, myelography with high-resolution CT should be the supplementary study. If surgery is planned, intraoperative spinal US should be used.

CT-guided fine needle aspiration of a periaortic collection.

Patients with infected aortic grafts suffer considerable morbidity and mortality. Therefore, it is important to make the diagnosis of infection to provide a reasonable chance for survival. Clinical signs of infection are usually apparent in the postoperative period. If absent, the diagnosis becomes more difficult. CT scanning is beneficial in detecting periaortic fluid and gas collections; however, it cannot differentiate postoperative gas from infection in all cases. This is especially true during the first 2 weeks after operation. CT-guided fine needle aspiration can provide a safe and reliable means to answer this clinical question.

Portal venous gas following a barium enema in a patient with Crohn's colitis. A benign finding.

Hepatic portal venous gas occurring during an air-contrast barium-enema examination in patients with inflammatory bowel disease is a benign finding. Patients with chronic ulcerative colitis have experienced some morbidity, while those with Crohn's colitis have not. It may not be necessary to treat all of these patients with antibiotics, especially asymptomatic patients with Crohn's colitis.