Inhibition of aromatase activity in human placental microsomes by 13-retro-antiprogestins.

Mifepristone (RU 486), used clinically for the termination of early pregnancy, and its acetyl and 13-retro (13 alpha) analogs show potent antiproliferative effects against estrogen-dependent human breast tumors and endometriosis. However, there has been no report on direct inhibition of aromatase by antiprogesterones. Aromatase inhibitors have been shown to be effective against estrogen-dependent breast cancer.

Tobacco alkaloid derivatives as inhibitors of breast cancer aromatase.

The inhibition of estrogen biosynthesis by the use of aromatase inhibitors is emerging as a valuable approach to breast cancer therapy. Because smoking has a profound effect on estrogen-related processes we examined the ability of tobacco constituents to suppress estrogen production by breast cancer aromatase. N-n-octanoylnornicotine and N-(4-hydroxyundecanoyl) anabasine suppressed aromatase activity in culture of two human breast cancer cell lines, MDA-MB-231 (IC50 of 310 and 20 microM, respectively) and SK-BR-3 (IC50 of 450 and approximately 2 microM, respectively).

Catalytic efficiency of expressed aromatase following site-directed mutagenesis.

Mutant aromatase cytochrome P-450s, expressed in CHO cells after transfection with cDNAs, have been characterized in terms of their catalytic efficiencies. After solubilization from microsomes, specific aromatase P-450 content of wild-type and mutants Pro308Phe, Asp309Asn, Asp309Ala and Phe406Arg was quantitated by a sandwich enzyme-linked immunosorbent assay (ELISA). Microsomal aromatase activity was determined by the 3H-water method using [1 beta-3H]androstenedione as substrate.

Competitive product inhibition of aromatase by natural estrogens.

In order to better understand the function of aromatase, we carried out kinetic analyses to assess the ability of natural estrogens, estrone (E1), estradiol (E2), 16 alpha-OHE1, and estriol (E3), to inhibit aromatization. Human placental microsomes (50 micrograms protein) were incubated for 5 min at 37 degrees C with [1 beta-3H]testosterone (1.24 x 10(3) dpm 3H/ng, 35-150 nM) or [1 beta-3H,4-14C]androstenedione (3.

Multiple functions of aromatase and the active site structure; aromatase is the placental estrogen 2-hydroxylase.

Androgen aromatase was found to also be estrogen 2-hydroxylase. The substrate specificity among androgens and estrogens and multiplicity of aromatase reactions were further studied. Through purification of human placental microsomal cytochrome P-450 by monoclonal antibody-based immunoaffinity chromatography and gradient elution on hydroxyapatite, aromatase and estradiol 2-hydroxylase activities were co-purified into a single band cytochrome P-450 with approx.

Kinetic properties of aromatase mutants Pro308Phe, Asp309Asn, and Asp309Ala and their interactions with aromatase inhibitors.

Mutant forms of aromatase cytochrome P-450 bearing modifications of amino acid residues Pro308 and Asp309 and expressed in transfected Chinese hamster ovary cells were subjected to kinetic analysis and inhibition studies. The Km for androstenedione for expressed wild type (11.0 +/- 0.

Purification of human placental aromatase cytochrome P-450 with monoclonal antibody and its characterization.

A simple and efficient method is described for the purification of microsomal aromatase cytochrome P-450 from human placenta. The enzyme was solubilized with Emulgen 913 and sodium cholate and subjected to chromatography on a column of Sepharose 4B coupled with a specific monoclonal antibody, followed by hydroxyapatite column chromatography. The specific cytochrome P-450 content of purified aromatase was 13.

Competitive inhibition of human placental aromatase by N-n-octanoylnornicotine and other nornicotine derivatives.

In a study of the effect of N-n-octanoylnornicotine and other acyl derivatives of nornicotine on the aromatization of androstenedione by human placental microsomal aromatase, we found that N-n-octanoylnornicotine, a component of cigarette smoke, exhibited competitive inhibition with an apparent Ki of 0.65 microM. This is comparable to that of aminoglutethimide, the clinically-used non-steroidal aromatase inhibitor.

Serum level of 19-hydroxyandrostenedione during pregnancy and at delivery determined by gas chromatography/mass spectrometry.

19-Hydroxyandrostenedione (19-OHA) is secreted from the adrenal glands in men and women and also from the placenta during pregnancy. It has been found to cause hypertension in animal models. We have synthesized [7,7-2H2]-19-OHA with high deuterium content and, together with [7,7-2H2]A and [9,11-2H2]estrone (E1), have developed a quantitative assay of serum level 19-OHA, A, and E1 using the gas chromatography/mass spectrometry-mass fragmentography method to monitor individual subjects throughout pregnancy.

Aromatase inhibitors in cigarette smoke, tobacco leaves and other plants.

A chance observation that cigarette smoke interferes with the aromatase assay led us to investigate tobacco leaf and smoke extracts for the presence of aromatase inhibitors. The highest inhibitory activity was found in the basic fraction of cigarette smoke. Further purification of this fraction led to the identification of N-n-octanoylnornicotine.