Depression enhanced the production of autoantibodies against 16α­hydroxyestrone-estrogen receptor adduct in breast cancer.

Mentally depressed breast cancer (MDBC) patients expressed estrogen receptor (ER) and 16α­hydroxyestrone (16α­OHE1) is directly responsible for causing breast cancer (BC). This study aimed to identify whether depression in breast cancer patients enhanced the production of autoantibodies against 16α­OHE1-ER adduct in breast cancer patients. The antibodies in the serum of 65 breast cancer patients (including 35 MDBC) and 40 control subjects were screened by direct binding, inhibition enzyme-linked immunosorbent assay (ELISA), and quantitative precipitin titration. Competition ELISA was also utilized for the estimation of 16α­OHE1 in the serum of 30 cancer patients. Autoantibodies from MDBC showed strong recognition to 16α­OHE1-ER in comparison to overall breast cancer patients (p < 0.05) and control subjects (p < 0.001). Although breast cancer sera showed high binding to 16α­OHE1-ER in comparison to ER (p < 0.05) or 16α­OHE1 (p < 0.001), the relative affinities of autoantibodies for 16α­OHE1-ER were found to be 1.38 × 10-7 and 1.23 × 10-7 for breast cancer and MDBC patients respectively. No significant difference, either in the level of 16α­OHE1 or 2­hydroxyestrone/16α­OHE1 ratio, was observed in the serum of cancer patients compared with controls, although inflammatory cytokines (IL-6, TNF-α) were significantly high in these patients. Depression in breast cancer patients augments the production of autoantibodies against 16α­OHE1-ER through the generation of inflammatory conditions. Depression in these patients increased the release of pro-inflammatory cytokines that generate more autoantibodies and show strong binding with 16α­OHE1-ER.

Detection of 16α-Hydroxyestrone-histone 1 Adduct as High-Affinity Antigen for Rheumatoid Arthritis Autoantibodies.

Increased concentrations of 16α-hydroxyestrone (16α-OHE1) have been observed in rheumatoid arthritis (RA), but the underlying mechanism of this remains elusive. Here we aimed to identify the role played by 16α-OHE1 in RA. In 40 RA patients, the specificities of antibodies from the sera of these patients were checked by direct binding, inhibition ELISA, and quantitative precipitation titration. Competition ELISA was also used for the estimation of 16α-OHE1 in the serum of different RA patients. RA IgG from a patient's sera showed strong recognition to 16α-OHE1-H1 (histone 1) adduct in comparison to control subjects (p < 0.001), as the formation of this adduct brings out various biochemical changes that might generate neo-epitopes, which have been well-recognized by these antibodies. The affinity of RA antibodies for 16α-OHE1-H1 (1.10 × 10- 7 M) was high, as detected by the Langmuir plot. Comparing RA patients to the controls, no significant differences were detected in the level of 16α-OHE1 or 2-hydroxyestrone/16α-OHE1 ratio. 16α-OHE1-H1 might have an antigenic role and function as a high-affinity antigen for RA autoantibodies and, therefore, could be used as a biomarker for this disease.

Potential biomarker for early risk assessment of prostate cancer.

BACKGROUND: Catechol estrogen quinones (CEQ) derived from 4-hydroxyestrone (4-OHE1) and 4-hydroxyestradiol (4-OHE2) react with DNA to form depurinating--N7Gua and--N3Ade adducts. This damage leads to mutations that can initiate breast and prostate cancer. To determine whether this damage occurs in humans, urine samples from men with prostate cancer and benign urological conditions, and healthy controls were analyzed. The objective was determining whether any of the cancer patients had formed the depurinating 4-OHE1(E2)-1-N3Ade adducts. METHODS: The adducts were extracted from samples by using affinity columns equipped with a monoclonal antibody developed for detecting 4-OHE1(E2)-1-N3Ade adducts. Eluted extracts were separated by capillary electrophoresis with field-amplified sample stacking and/or ultraperformance liquid chromatography. Absorption/luminescence spectroscopies and mass spectrometry were used to identify the adducts. RESULTS: 4-OHE1-1-N3Ade was detected at higher levels in samples from subjects with prostate cancer (n = 7) and benign urological conditions (n = 4) compared to healthy males (n = 5). CONCLUSION: This is the first demonstration that CEQ-derived DNA adducts are present in urine samples from subjects with prostate cancer.

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.

Synovial fluid estrogens in rheumatoid arthritis.

Experimental and clinical evidence suggest that immune reactivity is modulated by gender. Immune reactivity is greater in females than in males and lymphocytes and monocytes from female subjects shows higher antigen presenting activity and mitogenic responses. Steroid hormones can be converted along defined pathways to downstream hormones in the periphery. The conversion of dehydroepiandrosterone (DHEA) in target macrophages leads to an increase of downstream effector hormones (including estrogens), which may be an important factor for local immunomodulation at least in RA synovitis. The presence in the RA synovial fluids (SF) of an altered sex hormone balance resulting in lower immunosuppressive androgens and higher immunoenhancing estrogens, might determine a favorable condition for the development of the immuno-mediated RA synovitis and synovial hyperplasia. The increased estrogen concentration observed in RA SF of both sexes are characterized by the hydroxylated forms, in particular 16alpha-hydroxyestrone, that is a mitogenic and proliferative endogenous hormone. In contrast to 16alpha-hydroxylated estrogens, the 2-hydroxylated forms inhibit growth promoting effects of E2 and were found low in RA SF. Therefore, dose-related conversion to pro- or anti-inflammatory downstream metabolites of estrogens might support the dual role of estrogens (pro or anti-inflammatory) for example during estrogen replacement therapy, depending on local concentration (i.e. SF in RA) of 16alpha-hydroxyestrone or 2-hydroxyestrogens.

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.

Radioimmunoassay of 2-hydroxyesterone using antisera raised against antigenic complexes obtained by convenient methods.

Antigenic complexes of 2-hydroxyestrone (2-OHE1) were obtained by Mannich reaction of 2-OHE1 and bovine serum albumin (BSA) and by coupling of 2-OHE1 1-glutathione thioether to BSA using glutaraldehyde. Antiserum raised against the antigen obtained by the Mannich reaction had high affinity (Kd = 3.8 x 10(9) M-1) and relatively high specificity; cross reactivities for estrone, 4-hydroxyestrone and 2-methoxyestrone were 2.1%, 10% and 1.5%, respectively. The other antiserum also had high affinity (4.5 x 10(9) M-1) but its cross reactivities for the above three steroids were more than 100%. Concentrations of 2-hydroxyestrone in human plasma were determined by radioimmunoassay with the more specific antiserum and Sephadex LH-20 chromatography to be less than a minimum detectable amount (less than 10 pg/ml) (men), 20.9 pg/ml (women, proliferation) and 26.0 pg/ml (women, periovulation).

Inhibition of the preovulatory LH surge after a catecholestrogen (2-hydroxyestrone) antiserum injection in the third ventricle of cycling female rats.

Administration of a catecholestrogen (2-hydroxyestrone, 2-OHE1) antiserum (2-OHE1-AS) in the third ventricle of cycling female rats, on the morning of proestrus, leads to a significant reduction in the afternoon LH surge. These responses are dose-dependent and can be observed even when the 2-OHE1-AS is injected on the diestrus morning. Almost similar results were obtained with an antiserum against 17 beta-estradiol (17 beta-E2). Nevertheless, the fact that the central immunoneutralization of 2-OHE1 impedes the preovulatory surge of LH at a time of high peripheral levels of 17 beta-E2 strengthens the idea of a specific role for 2-OHE1 in the control of cycling LH release.

Anti-oestrogen antibodies in users of oral contraceptives and in patients with systemic lupus erythematosus.

Recent studies have demonstrated that many patients with SLE have elevated plasma levels of the minor oestrogen metabolite 16 alpha-hydroxyestrone (16 alpha OHE). This oestrogen is unique in its ability to react with lysine residues and form stable, covalent Heyns products with proteins. Increased levels of 16 alpha OHE-modified proteins have been found to occur on the membranes of red cells and lymphocytes in patients with SLE. In the present study, patient and control sera were analysed for the presence of circulating immunoglobulins which react with an oestrogen hapten. Anti-oestrogen antibodies were detected in 26% (9/34) of male and female SLE patients, and were found to correlate both with levels of plasma 16 alpha OHE (P less than 0.001) and with the presence of active disease (P less than 0.005). Surprisingly, this antibody activity was also observed in 25% (13/52) of normal, disease-free women who had a history of oral contraceptive use. No detectable activity was observed in normal men, women who had not taken oral contraceptives, or patients with a variety of other immunological diseases. The possible role of anti-oestrogen antibodies in both the hormonal exacerbation of SLE and in the long-term sequelae of oral contraceptive usage is discussed.

Characterization of antisera to the addition product formed by the nonenzymatic reaction of 16 alpha-hydroxyestrone with albumin.

16 alpha-Hydroxyestrone (16 alpha OHE) has been shown previously to react nonenzymatically with proteins via a Heyns rearrangement of a Schiff base intermediate. Albumin modified by the addition of 16 alpha OHE is immunogenic, despite a relatively low molar substitution. High-titre antisera can be elicited which have a very high affinity toward the estrogen hapten. Cross-reactivity analysis reveals a high specificity for the phenolic A-ring and a lack of specificity to chemical substituents in the D-ring, the site of covalent linkage. The antisera reacts equally well with 16 alpha OHE-peptides as with 16 alpha OHE-lysine, suggesting the utility of this antisera in analyzing either enzymatically or chemically hydrolyzed proteins for the presence of 16 alpha OHE adducts. Immunochemical analysis of proteins modified by 16 alpha OHE may provide insight into the pathogenesis of systemic lupus erythematosus, an autoimmune disease in which elevated levels of 16 alpha OHE are known to occur.

Synthesis of immunogenic C-6 derivatives of 2-methoxyestrone and 2-methoxyestradiol-17 beta and characterization of the corresponding antisera.

The synthesis, purification and structural confirmation of the 6-(carboxymethoxyimino) derivatives of 2-methoxyestrone and 2-methoxyestradiol-17 beta are described. These derivatives were coupled to bovine serum albumin by the mixed anhydride method, and rabbits were immunized with the product. The resulting antisera showed high affinity and specificity for 2-methoxyestrone and 2-methoxyestradiol-17 beta, respectively, with low cross reactivities to structurally related estrogens.

Low plasma levels of 2-hydroxyestrone are consistent with its rapid metabolic clearance.

Published plasma levels of the catechol estrogen 2-hydroxyestrone (2-OHE1) are comparable to those of estrone and estradiol. In light of the very high (40,000 L/d) metabolic clearance rate of 2-OHE1, these concentrations imply unreasonable production rates. We therefore re-examined plasma 2-OHE1 levels using a modified radioimmunoassay procedure. Plasma samples are extracted with ethyl acetate and passed over a short column of LH-20 Sephadex before equilibration with an antiserum directed against a 2-hydroxyestrone-17-(O-carboxymethyl)oxime-bovine serum albumin conjugate. Plasma 2-OHE1 concentrations are indistinguishable from blank (< 15 pg/ml) in men and non-pregnant women, but rise to approximately 200 pg/ml during pregnancy. These values for 2-OHE1 levels are consistent with the rapid metabolic clearance of this catechol estrogen.

A radioimmunoassay method for urinary catechol estrogens.

A radioimmunoassay method for urinary catechol estrogens is described; The specific nature of the antisera allows direct analyses of acid hydrolyzed urine. A LH-20 Sephadex column chromatography can be employed for individual determinations of 2-hydroxyestrone and 2-hydroxyestradiol. The excretion of catechol estrogens during menstrual cycles ranged from 14.48 to 50.15 microgram per 24 hours, whereas, during the last trimester of pregnancies, the values ranged from 129.30 to 1758. 20 microgram per 24 hours.