Development of monoclonal antibodies to 4-hydroxyestrogen-2-N-acetylcysteine conjugates: immunoaffinity and spectroscopic studies.

Catechol estrogen quinones (CEQ) derived from oxidation of the catechol estrogens 4-hydroxyestrone (4-OHE1) and 4-hydroxyestradiol (4-OHE2) can conjugate with glutathione (GSH), a reaction that prevents damage to DNA and can provide biomarkers of exposure to CEQs. Monoclonal antibodies (MAb) to 4-OHE1(E2)-2-N-acetylcysteine [4-OHE1(E2)-2-NAcCys] were developed and characterized by immunological and spectroscopic studies. The NAcCys conjugate is the hydrolytic product of the corresponding conjugate with GSH, followed by N-acetylation of cysteine. MAbs were produced by immunizing mice with 4-OHE1(E2)-2-NAcCys attached to an appropriate linker that was conjugated to keyhole limpet hemocyanin (KLH). Hybridoma cell lines were screened using 4-OHE1(E2)-2-NAcCys conjugated to ovalbumin (OA). There is no immunological cross-reactivity between KLH and OA. Hence, positive hybridoma cell lines secreting antibody against 4-OHE1(E2)-2-NAcCys could be rapidly identified using OA-4-OHE1(E2)-2-NAcCys. An affinity column was developed and used to purify MAb against 4-OHE1(E2)-2-NAcCys. The purified MAb was immobilized on an agarose bead column. This column was used to capture and preconcentrate the hapten of interest out of urine samples. A number of structurally related standards were used to estimate the selectivity and specificity of the chosen MAb. Capillary electrophoresis (CE) with field-amplified sample stacking in absorbance detection mode and laser-induced low temperature luminescence measurements were used to identify and quantitate the 4-OHE1(E2)-2-NAcCys conjugates and related compounds released from the affinity column. Femtomole detection limits have been demonstrated. Future prospects in clinical diagnostics for testing human exposure to CEQ by urine analysis are briefly addressed.

Estrogen receptor-independent catechol estrogen binding activity: protein binding studies in wild-type, Estrogen receptor-alpha KO, and aromatase KO mice tissues.

Primary evidence for novel estrogen signaling pathways is based upon well-documented estrogenic responses not inhibited by estrogen receptor antagonists. In addition to 17beta-E2, the catechol estrogen 4-hydroxyestradiol (4OHE2) has been shown to elicit biological responses independent of classical estrogen receptors in estrogen receptor-alpha knockout (ERalphaKO) mice. Consequently, our research was designed to biochemically characterize the protein(s) that could be mediating the biological effects of catechol estrogens using enzymatically synthesized, radiolabeled 4-hydroxyestrone (4OHE1) and 4OHE2. Scatchard analyses identified a single class of high-affinity (K(d) approximately 1.6 nM), saturable cytosolic binding sites in several ERalphaKO estrogen-responsive tissues. Specific catechol estrogen binding was competitively inhibited by unlabeled catechol estrogens, but not by 17beta-E2 or the estrogen receptor antagonist ICI 182,780. Tissue distribution studies indicated significant binding differences both within and among various tissues in wild-type, ERalphaKO, and aromatase knockout female mice. Ligand metabolism experiments revealed extensive metabolism of labeled catechol estrogen, suggesting that catechol estrogen metabolites were responsible for the specific binding. Collectively, our data provide compelling evidence for the interaction of catechol estrogen metabolites with a novel binding protein that exhibits high affinity, specificity, and selective tissue distribution. The extensive biochemical characterization of this binding protein indicates that this protein may be a receptor, and thus may mediate ERalpha/beta-independent effects of catechol estrogens and their metabolites.

Preparation of specific antisera to 15alpha-hydroxyestrogens.

The synthesis of haptens of 15alpha-hydroxyestrone, 15alpha-hydroxyestradiol, and 15alpha-hydroxyestriol (estetrol) was undertaken, to obtain specific antisera required for enzyme immunoassay. 3-(1-Carboxypropyl) ethers of these 15alpha-hydroxyestrogens were prepared and conjugated with bovine serum albumin and horseradish peroxidase. The specificity of antisera elicited against bovine serum albumin conjugates was checked by the enzyme immunoassay by using horseradish peroxidase-labeled antigen, and proved to be satisfactory in terms of cross-reactivities to related compounds.

New synthesis of delta 6-estrogens.

An efficient approach to synthesize delta 6-estrogens is described. The key steps in the synthesis are the introduction of a hydroxyl group at the C-6 position of a suitably protected estrogen using a superbase and subsequent dehydration with Martin sulfurane reagent or methyltriphenoxyphosphonium iodide. The two-step synthetic procedure readily gave the delta 6-estrogens in high yield.

Reactivation of human placental 17 beta, 20 alpha-hydroxysteroid dehydrogenase: affirmation of affinity labeling principles.

Human placental 17 beta, 20 alpha-hydroxysteroid dehydrogenase was completely inactivated by the affinity alkylator, 3-bromoacetoxy-1,3,5(10)-estratrien-17-one (estrone 3-bromoacetate). The inactivated enzyme was then reactivated to 100% of the enzyme activity by base-catalyzed hydrolysis of the steroidalester-enzyme conjugate. After the reactivated enzyme was repurified by dialysis, re-inactivation studies were performed on it. The reactivated enzyme could not be re-inactivated by the original alkylator, estrone 3-bromoacetate. However, 16 alpha-bromoacetoxyestradiol-17 beta 3-methyl ether caused a loss of reactivated enzyme activity at a rate comparable to that for the native enzyme. These observations demonstrate that a specific amino acid modification within the enzyme active site was produced by estrone 3-bromoacetate alkylation and suggest that the conformation of the active center was essentially unaltered. Thus, these successful reactivation studies of 17 beta, 20 alpha-hydroxysteroid dehydrogenase affirm the specificity of affinity labeling. This methodology also offers a new tool to investigate the steroid binding regions of macromolecular proteins.

A short efficient synthesis of 16-oxygenated estratriene 3-sulfates.

A novel synthesis of sodium 17-oxo-16 alpha-hydroxy-1,3,5(10)-estratrien-3-yl sulfate (4), sodium 16 alpha, 16 beta-dihydroxy-1,3,5(10)-estratrien-3-yl sulfate (5) and sodium 16-oxo-17 beta-hydroxy-1,3,5(10)-estratrien-3-yl sulfate (6) is described. 16 alpha-Bromo-3-hydroxy-1,3,5(10)-estratrien-17-one (1) was efficiently synthesized in one step with 70-97% yield by bromination of 3-hydroxy-1,3,5(10)-estratrien-17-one with cupric bromide. 3,16 alpha-Dihydroxy-1,3,5(10)-estratrien-17-one (3) was quantitatively obtained by controlled stereospecific hydrolysis of the bromoketone 1 with sodium hydroxide in aqueous pyridine. The bromoketone 1 was converted to the 16 alpha-hydroxy-17-ketone 3-sulfate 4 by sulfation with chlorosulfonic acid in pyridine and a subsequent controlled hydrolysis in a high yield without formation of the other ketols. Treatment of the sulfate 4 with sodium borohydride have the triol sulfate 5. The sulfate 4 was also rearranged to the 17 beta-hydroxy-16-ketone 6 with sodium hydroxide in water in a quantitative yield.

Structure-activity relationships of four 11-hydroxyestrones isomeric at the C-9 and C-11 positions.

The synthesis of 11 alpha-hydroxyestrone, 11 alpha-hydroxy-9 beta-estrone, and 11 beta-hydroxy-9 beta-estrone are presented. The reduction of 11-keto-9 beta-estrone 17-ethyleneketal by sodium in ethanol or sodium borohydride resulted in 11-hydroxy-9 beta-estrones. The 11-hydroxyl group configurations were opposite to expectations: sodium in boiling ethanol afforded the axial 11 beta-hydroxy-9 beta-estrone, while sodium borohydride in boiling tetrahydrofuran gave the equatorial 11 alpha-hydroxy-9 beta-estrone. In immature rat uterotropic bioassays using subcutaneous injections, 11 alpha-hydroxyestrone was 2 times as active as 11 alpha-hydroxy-9 beta-estrone, and 11 beta-hydroxyestrone was 10 times as active as 11 beta-hydroxy-9 beta-estrone.