[Association of polymorph variants of CYP1A2 and CYP1A1 genes with reproductive and thyroid diseases in female workers of petrochemical industry].

The article presents results obtained in study of relationship between polymorph variants of CYP1A1 and CYP1A2 genes with reproductive and thyroid diseases risk in female workers of petrochemical industry, when compared with reference group females. Variants TD and DD of CYP1A2 gene appeared to be associated with nodes formation in uterus and breast in female workers and reference group females. Following liability markers are obtained: homozygous in rare allele genotype CC of CYP1A1 gene for reproductive and thyroid diseaes (fibrous cystic mastopathy and nodular goitre), heterozygous genotype AG of CYP1A1 gene in uterine myoma and fibrous cystic mastopathy, homozygous in deleted T genotype of CYP1A2 gene in autoimmune thyroiditis. Occupational hazards and long length of service at hazardous industries increase effects of rare alleles of the genes studied.

Prostate-specific antigen gene expression and telomerase activity in breast cancer patients: possible relationship to steroid hormone receptors.

Breast cancer, the most prevalent cancer among women, is a steroid hormone receptor-dependent cancer. Recently, it has been shown that telomerase and prostate-specific antigen (PSA) gene expressions are under control of steroid hormone receptors. The aim of this study was to investigate the relationship between telomerase activity and PSA gene expression with steroid hormone receptors in breast cancer patients. This study consisted of 50 women with breast benign tumors and 50 malignant (invasive) tumors. Telomerase activity was measured in tumor cytosol of samples by telomeric repeat amplification protocol (TRAP) assay. PSA protein and its mRNA expression were measured using ultrasensitive immunoassay and RT-PCR technique in all tumor tissues, respectively. Estrogen and progesterone receptors were stained using immunohistochemistry in tumor tissues. Telomerase activity was detected in all of the invasive breast cancer tissues. The difference of relative telomerase activity (RTA) values between stages and grades were statistically significant (p < 0.05). The PSA mRNA was detected only in benign tumors and stage I and grade I malignant tumor cytosol. Difference of tumor cytosol PSA levels between the cases and control groups and also between all grades and stages of diseases were significant (p < 0.05). There was an inverse significant correlation between the RTA and PSA protein levels in the case groups (r = -0.42, p < 0.05). There was a statistically significant difference between ER/PR with PSA level and telomerase activity in tumor tissues (p < 0.05). It is speculated that differential expression of PSA and telomerase genes in breast tumors are under control of steroid hormone receptors and could be used as a target for treatment in the future.

Cathepsin-D expression in breast lesion: an immunohistochemical study.

Cathepsin-D (CathD) is an aspartyl lysosomal protease expressed in all tissues that might play a role in antigen processing, cell proliferation and tissue renewal, and activation of different pro hormones. The aim of our study was to compare the expression of CathD in most common breast tumors and tumor-like breast lesions. The study includes 21 patients with histologically verified breast lesions (adenosis, ductal hyperplasia, fibroadenomas, and different types of invasive carcinoma). We investigated the cathepsin-D expression in these breast lesions using immunohistochemistry (IH; paraffin-embedded tissues). Cathepsin-D staining within each lesion was assessed by estimating the area of the objects and the medium pixel intensity per object, as the integrated optical density (IOD). The immunostaining was more obvious in breast invasive carcinomas and macrophages. The reaction in tumor tissue was heterogeneous with little variation of staining intensity in positive tumor cells. Adenosis had the maximum area/signal intensity from all studied breast benign lesions (p<0.001, Student t-test). The general tendency (all benign lesions, lobular carcinomas and G3 ductal invasive carcinoma) was a more prominent representation of the cellular compartment. In the G3 ductal invasive carcinoma-type, the group of patients with metastases had a stronger expression in the cellular compartment. These results suggest that CathD expression was strongest in malignant than in benign breast disease, the positivity being present in both epithelial neoplastic and stromal cells. We also conclude that our procedure in IOD measurement is prone to less subjective-related biases, and thus more accurate and constant than other methods employed by other authors.

Steroid sulfatase, arylsulfatases A and B, galactose-6-sulfatase, and iduronate sulfatase in mammary cells and effects of sulfated and non-sulfated estrogens on sulfatase activity.

Sulfatase enzymes have important roles in metabolism of steroid hormones and of glycosaminoglycans (GAGs). The activity of five sulfatase enzymes, including steroid sulfatase (STS; arylsulfatase C), arylsulfatase A (ASA; cerebroside sulfatase), arylsulfatase B (ASB; N-acetylgalactosamine-4-sulfatase), galactose-6-sulfatase (GALNS), and iduronate-2-sulfatase (IDS), was compared in six different mammary cell lines, including the malignant mammary cell lines MCF7, T47D, and HCC1937, the MCF10A cell line which is associated with fibrocystic disease, and in primary epithelial and myoepithelial cell lines established from reduction mammoplasty. The effects of estrogen hormones, including estrone, estradiol, estrone 3-sulfate, and estradiol sulfate on activity of these sulfatases were determined. The malignant cell lines MCF7 and T47D had markedly less activity of STS, ASB, ASA, and GAL6S, but not IDS. The primary myoepithelial cells had highest activity of STS and ASB, and the normal epithelial cells had highest activity of GALNS and ASA. Greater declines in sulfatase activity occurred in response to estrone and estradiol than sulfated estrogens. The study findings demonstrated marked variation in sulfatase activity and in effects of exogenous estrogens on sulfatase activity among the different mammary cell types.

Enzyme activities in normal, dysplastic, and cancerous human breast tissues.

The activities of six different enzymes were compared in 29 normal, 34 dysplastic, and 80 cancerous (both primary and metastatic) human breast tissues; in MCF-7 cells; and in primary rat mammary tumors. Benign lesions generally showed enzyme activities similar to those of normal breast tissues. Malignant tumors had significantly increased activities of lactate dehydrogenase (LDH), malate dehydrogenase (MDH), fructose-bisphosphate aldolase, hexokinase (HK), pyruvate kinase (PK), and creatine kinase. Enzyme activity in the malignant tumor was always higher than that in apparently normal or fibrocystic tissue from the same patient. Enzyme activities did not correlate with the levels of estrogen and progesterone receptors. LDH, MDH, and HK were elevated to a similar extent in all the tissues examined. Conversely, PK was elevated to a much greater extent in cancerous tissues, particularly in MCF-7 cells. The elevated activities of these enzymes may have diagnostic potential, especially when tumor tissue and apparently normal tissue from the same patient are compared.

Proteases in cyst fluid from human gross cyst breast disease.

Cyst fluid from women with gross cystic breast disease was found to contain protease activity when assayed against [14C]albumin. At least six different proteases were detected when the fluid was fractionated by a combination of S-300 Sephacel, hydroxylapatite, and DEAE-Sephacel chromatographic techniques. The distribution of the proteases appeared to be related to the ionic composition of the fluids. A major protease component, found in both high Na and high K fluids, was isolated. It showed chymotryptic cleavage characteristics against the beta-chain of insulin. It was partially inhibited by alpha 2-macroglobulin, N-tosyl-L-phenylalanine chloromethyl ketone, and benzamidine but not by leupeptin, pepstatin, N-tosyl-L-lysine chloromethyl ketone, or alpha 1-protease inhibitor. The protease has an apparent molecular weight of 110,000 with Mr 24,000 subunits. This protease may be identical or closely associated with Haagensen's GCDFP-24 progesterone binding protein which was isolated in a similar manner. An imbalance between protease and protease inhibitors in cyst fluid may account for gross cyst formation and may be involved in the tumorigenic process. The accumulation of poorly diffusible peptide fragments, as a result of protease activity, would increase the oncotic pressure leading to enlargement of the cyst cavity as water enters to reestablish osmotic equilibrium.

Isozyme patterns of normal, benign, and malignant human breast tissues.

Isozyme patterns of 23 different enzymes were compared in normal, benign, and malignant breast tissues; in MCF-7 cells; and in organoids of normal human breast tissue. Benign lesions generally showed isozyme patterns similar to those of normal tissues. Lactate dehydrogenase isozyme 5 was significantly increased in malignant tumors; MCF-7 cells had only lactate dehydrogenase (L-lactate:NAD oxidoreductase; EC 1.1.1.27). The mitochondrial form of malate dehydrogenase was also significantly increased in human malignant tumors; this was especially evident when comparing tumor and apparently uninvolved breast tissue from the same patient. The K4 isozyme of pyruvate kinase was the major form in most malignant breast tumors, but in only 41% of normal tissues, 30% of fibrocystic disease specimens, and 46% of fibroadenomas. A more anodal band of pyruvate kinase, probably a K3M or K3Kpm hybrid, predominated in most normal and benign tissues, but in only 63% of primary and 56% of secondary tumors. All specimens had predominantly creatine kinase BB, aldolase A4, and hexokinase I. Traces of aldolase A3C and of hexokinase II were observed in some tumors. None of the tumors had the Regan variant of alkaline phosphatase. The isozymes of lactate and malate dehydrogenases and of pyruvate kinase appear to be the most promising as putative tumor markers.

Demonstration of human breast carcinoma cells in cryosections and primary monolayer cultures of surgical biopsies by neotetrazolium reductase cytochemistry.

The present study describes a cytochemical approach to demonstrate human breast carcinoma cells in cryosections and in primary monolayer cultures from surgical biopsies. The material consisted of biopsies from 52 carcinomas and 29 benign lesions. Cryosections and cultures were incubated to demonstrate NADPH-neotetrazolium reductase in an atmosphere of 99.5% oxygen. Incubation time and section thickness were adjusted to accomplish the same level of reaction in cells of cryosections and corresponding cultures. Positive reaction was thus confined to epithelial elements and to the wall of some smaller blood vessels. More than one-half of the carcinoma cells showed moderate to strong reaction in cryosections from 29 of 52 carcinomas whereas no reaction was seen in ductules of normal appearance adjacent to these carcinoma cells. Positive reaction was seen in epithelial cell islets in primary cultures of 16 of the 40 carcinomas cultured. In cryosections from fibroadenomas and fibrocystic disease specimens only apocrine metaplasia consistently showed positive reactions compared with less than 10% of other ductular profiles in a given cryosection. This pattern of reaction was reflected in the derived primary cultures in which positive reaction was found in epithelial cell islets in only one of 19 biopsies cultured. The presence of human milk fat globule membrane antigen was used to demonstrate the epithelial nature of the cell islets seen in cultures of biopsies from both benign lesions and carcinomas. NADPH-neotetrazolium reductase positive islets from carcinoma biopsies were frequently aneuploid whereas most negative islets from carcinoma biopsies were diploid as were all islets from benign tissues.

Purification and properties of an esterase from human breast cyst fluid.

Levels of estradiol 17 beta-ester hydrolytic activity in the breast cyst fluid (BCF) from 25 different women with fibrocystic disease of the breast were found to vary over a wide range (0-2.4 nmol/min/mg protein for estradiol acetate). On the basis of electrophoretic mobility on agarose gels, the activity from different individuals appeared to be identical. The esterase activity from a single BCF sample was purified to near homogeneity by differential ammonium sulfate precipitation, ion-exchange, and hydrophobic interaction chromatography. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis, after the final purification step, showed two bands with molecular weights of approximately 22,000 and 23,000, neither of which was immunoreactive with a rabbit antibody raised to a crude esterase-free BCF preparation. Esterase activity could be demonstrated after extraction and renaturation of the protein eluted from the Mr 22,000 band. Resolution of the gel, however, was not good enough to rule out the presence of esterase activity in the Mr 23,000 protein. High performance liquid chromatography gel exclusion chromatography indicated a molecular weight of 90,000-95,000 for the esterase activity in crude BCF and approximately 225,000 for the purified activity, suggesting the native protein to be a tetramer which aggregated during purification. Although the natural substrate of the BCF esterase is unknown, the enzyme is able to cleave a variety of esters including acetate, valerate, and stearate esters of estradiol and p-nitrophenyl hexanoate. It is completely inhibited by diisopropylflurophosphate and diethylnitrophenyl phosphate and partially inhibited by NaF and ebelactone. The substrate and inhibitor profile of the enzyme indicates that it is a "B"-type carboxylesterase and not a protease. A comparison of the properties of the BCF esterase with those of esterases from the formed elements of the blood or from plasma suggests that the BCF esterase is not of blood origin and is probably derived from the cyst itself. Physiologically inactive lipoidal estrogens have been shown to be present in many human body fluids and tissues and it is possible that these esters serve as storage forms of the active hormone in hormonally sensitive tissues where the free steroid could be regenerated by hydrolysis.

Quantification and molecular analysis of cathepsin D in breast cyst fluids.

Cyst fluids from 55 premenopausal women with gross cystic breast disease were classified by K+/Na+ ratio: 19 with ratio over 1 (type I) and 36 with ratio less than 1 (type II). Immunoradiometric assay of cathepsin D in both types of cyst fluids revealed the presence of large amounts of this proteinase. The average concentration of cathepsin D in type I cyst fluids was 63.3 nmol/l, which was significantly higher than that corresponding to type II cyst fluids (35.1 nmol/l). Immunoprecipitation analysis of intracystic cathepsin D demonstrated that this protein was present as the 52 kD non-processed precursor form of the molecule. Since procathepsin D is a useful prognostic marker in breast carcinoma, we suggest that cyst fluid quantification of cathepsin D could aid to detect patients affecting of gross cystic disease with higher risk for developing breast cancer.

Quantitative cytochemistry of glucose-6-phosphate dehydrogenase in benign and malignant breast tumours.

Glucose-6-phosphate dehydrogenase (G6PD) activity was quantified cytochemically in mammary epithelial cells within frozen tissue sections from 38 patients with breast cancer and 44 with benign breast disease. G6PD activities were measured under atmospheres of both N2 and O2. The mean (S.E.) G6PD value 2.5 (0.23) IE U/min measured in N2 in mammary epithelial cells from the group of malignancies was significantly greater than that of 1.6 (0.37) IE U/min in the benign group (P less than 0.001), but there was considerable overlap between individual values. G6PD measured in O2 was detectable in 84% of malignancies compared to only 14% of benign biopsies and the group mean of 1.3 (0.18) IE U/min in the former was significantly greater than that of 0.35 (0.20) IE U/min in the latter (P less than 0.001). Significant correlations between G6PD activities measured in N2 and O2 were observed in both groups. The techniques present a sensitive method of identifying increases in G6PD activity in mammary epithelial cells and provide an assay that in a majority of cases permits the separation of malignant from benign tissues.

Glutathione peroxidase, glutathione S-transferase and glutathione reductase activities in normal and neoplastic human breast tissue.

Glutathione peroxidase (GSH-Px), glutathione S-transferase (GSH-Tr) and glutathione reductase (GSSG-Rx) activities have been determined in normal and neoplastic human breast tissues. Large interindividual variations in the activities of all enzymes tested were found in both tumor and non-tumor specimens. In general a significant increase in the activities of the 3 enzymes was found in tumors, whereas in fibroadenoma they were as high as in healthy tissues. When a comparison was made between normal and neoplastic tissues of the same individual, GSH-Tr and GSSG-Rx activities were found to be higher in 15 and 11 cases, respectively, out of 17. GSG-Px activity was higher in all cases. From measurement of GSG-Px activity with both H202 and cumene hydroperoxide, it was deduced that human breast contains only the selenium-dependent form.

Effect of breast cyst fluid on oestrogen 17-oxidoreductase activity in MCF-7 breast cancer cells.

Breast cyst fluid (BCF) was found to stimulate oestrogen 17-oxidoreductase activity in the reductive direction, i.e., conversion of oestrone (E1) to oestradiol (E2), in MCF-7 breast cancer cells. Dialysis of BCF revealed that this property of BCF was present in both dialysed BCF and dialysate, implying that both high and low mol. wt. substances were responsible for stimulating E1 to E2 conversion. Gel filtration of dialysed BCF revealed that the high mol. wt. substances responsible for the stimulation of E1 to E2 conversion had mol. wts. of approximately 11 kD and 68 kD. This property of BCF would serve to increase the concentration of E2, a steroid which may play a role in mammary carcinogenesis.

Immunolocalization of aromatase and other steroidogenic enzymes in human breast disorders.

Numerous studies have shown that human breast cancer tissue has the potential to synthesize estrogen through aromatization, which may act as a local growth factor of hormone-dependent cancer cells. This study was performed to localize the site of aromatization in human breast disorders by immunohistochemistry and correlate the findings with steroid receptors, clinicopathological findings, and other steroidogenic enzymes. Specimens from 60 cases of breast disorders, including 33 cases of breast cancer and 27 cases of benign proliferative disorders, were studied immunohistochemically for aromatase. In the carcinoma cases estrogen receptor (ER) and progesterone receptor (PgR) status was determined by enzyme immunoassay and immunohistochemistry, and other steroidogenic enzymes, including P450scc (side-chain cleavage), 3 beta HSD (hydroxysteroid dehydrogenase), and P450c17, were immunolocalized. Aromatase was immunolocalized in interstitial cells and adipocytes as well as other cells in both benign and malignant breast tissues. However, strong immunoreactivity was observed in adipocytes adjacent to carcinoma in all carcinoma cases and in interstitial or stromal cells around carcinomatous glands in 20 carcinoma cases. Intratumoral staining for aromatase did not correlate significantly with age, clinical stage, histopathological type, lymph nodes metastasis, or ER and PgR status. P450scc and 3 beta HSD were focally observed in 18 and 12 cases of carcinoma, respectively, but P450c17 was never observed. Aromatase expression in stromal or interstitial cells, including adipocytes, in breast cancer may be induced by carcinoma cells and locally synthesized estrogens could function as paracrine hormones. Intratumoral aromatase in human breast neoplasms correlated with malignant phenotypes but not with ER status or prognostic parameters, suggesting that other synthetic systems probably generate any biologically significant locally synthesized estrogens in hormone-dependent breast malignancy.

Accumulation of 4- and 5-ene steroid sulfates in human breast cyst fluids.

Gross cystic disease of the breast is one of the most common diseases of adult females. Breast cyst fluid contains various steroid hormones. In order to obtain more information about the concentrations of 4- and 5-ene steroids in human breast cyst fluids, levels of pregnenolone sulfate (PREGS), pregnenolone (PREG), dehydroepiandrosterone sulfate (DHEAS) and dehydroepiandrosterone (DHEA) were determined by high-performance liquid chromatography (HPLC). A total of 35 human breast cyst fluid samples, obtained from 35 patients (28-54 years old) were analyzed. Cyst fluid electrolytes were simultaneously determined. Levels of PREGS (mean+/-S.D.) were 26.9+/-20.0 micromol/l (N=35) and of PREG were <0.1 micromol/l. Levels of DHEAS and DHEA were 89.1+/-111.7 micromol/l (N=35) and 0.3+/-0.2 micromol/l (N=35), respectively. Cyst fluids were divided into two groups (types I and II) according to their electrolyte ratio (K(+)/Na(+)). The cysts of the type I group (K(+)/Na(+) >1.5) contained significantly higher levels of PREGS (39.9+/-21.1 micromol/l) and DHEAS (133.2+/-87.9 micromol/l) than those of the type II group (K(+)/Na(+) <1.5), the mean levels of which were 19.8+/-16.2 micromol/dl for PREGS, and 36.3+/-29.0 micromol/dl for DHEAS (P<0.05). PREGS and DHEAS levels in the cysts were significantly correlated (r=0.49; P<0.01). Human breast cyst fluids contain high concentration of DHEAS and PREGS, especially in the cyst fluids containing high K(+)/Na(+) ratios.

Matrix-metalloproteinases 1, 2, and 3 and their tissue inhibitors 1 and 2 in benign and malignant breast lesions: an in situ hybridization study.

Invasive growth requires degradation of extracellular matrix. Altered expression of matrix degrading enzymes may indicate an increased potential for invasive growth. We determined the expression patterns of matrix-metalloproteinases (MMP)-1, -2, and -3 and of the tissue inhibitors of metalloproteinases (TIMP)-1 and -2 by in situ hybridization with isotopically labeled RNA probes in normal breast tissue (n=6), fibrocystic disease (n=20), five cases of which contained radial scars, lobular carcinoma in situ (CLIS; n=5), ductal carcinoma in situ (DCIS; n=9) and invasive carcinomas (n=24). Only a few cells displayed MMP-1- and MMP-2-specific labeling in normal breast tissue and fibrocystic disease. Noninvasive ductal carcinomas showed elevated MMP-2 transcript levels in peritumor stromal cells in the absence of significant MMP-1 specific signals. In general, compared with adjacent normal breast tissue, a gradual increase of MMP-2 was found in noninvasive to invasive cancers. Invasive ductal and lobular carcinomas displayed co-expression of MMP-1 and MMP-2 by stromal cells, mainly of the invasion front, with high signal intensity particularly in high-grade invasive carcinomas. Tumor cells and peritumor stroma showed low MMP-3 transcript levels, especially in medullary carcinomas. TIMP-1 and -2 transcript levels were increased in invasive carcinomas correlating with the histological grade. These RNA expression patterns suggest an increased invasive potential in breast carcinomas even prior to histologically overt invasive growth.

Cyst fluid proteases.

The precise origin of breast cyst fluid remains obscure. Molina has presented evidence that type II cysts (high Na/K ratio) may be transudative, that is, partly derived from plasma elements which enter through gap junctions, while Type I cysts (high K/Na ratio) are primarily secretory. In transudative cysts, plasma protease inhibitors may be present, but the balance between protease and its inhibitors may fluctuate as a result of as yet undetermined circumstances. An imbalance between the protease activity of cyst fluid and its inhibitors may be involved in the pathogenesis of breast gross cystic disease. Accumulation of protein fragments with resistant bonds would produce an elevated oncotic pressure causing a shift of fluid into the cyst capsule. Albumin is a good substrate for the protease, which may account for its low concentration in cyst fluid. The major protease fraction closely corresponds to the progesterone binding protein (GCDFP-24) described by Haagensen. Affinity columns containing aprotinin or benzamidine ligands retain the protease which can then be eluted with 0.5 M NaCl. The HD1 protease and progesterone binding protein are either tightly complexed or are the same protein. Cyst fluid is a complex mixture of biomolecules. If the progesterone binding protein is a protease, many questions must be answered concerning the influence of cyst fluid steroids, lipids, anions, and cations on enzyme action. Determination of the amino acid sequence of HD1 may help elucidate the source of the enzyme and its relationship to other tissue proteases. Human plasma contains inhibitors of this protease activity. When pooled, dialyzed plasma was mixed with pooled, dialyzed cyst fluid, the ratio of plasma/cyst fluid at which all activity was inhibited was 6/1. A comparison of the rate of cleavage of three 14C-protein substrates shows that cyst fluid proteases cleave in a characteristic manner, distinct from either trypsin or calpain. A simple method for semiquantitative estimation of protease activity in cyst fluid is described which utilizes prestained Coomassie blue-albumin containing agarose gel plates. All cyst fluids tested had protease activity but showed variability in their ability to cleave 14C-albumin by a factor of 4. There is much direct and indirect evidence that proteases are involved in the cancer process. In view of the higher than normal incidence of breast cancer in women who have had gross cystic breast disease, the possibility exists that an imbalance between these proteases and their inhibitors is somehow involved.

Immunofluorometric assay of human kallikrein 6 (zyme/protease M/neurosin) and preliminary clinical applications.

BACKGROUND: The human kallikrein gene family has contributed the best prostatic biomarkers currently available, including prostate-specific antigen (PSA) and human glandular kallikrein 2 (hK2). Recently, new members of the human kallikrein gene family have been identified. One new member is the KLK6 gene, encoding for human kallikrein 6 (hK6), which is also known as zyme/protease M/neurosin. In this paper, we describe development of antibodies and a sensitive immunofluorometric procedure for hK6 protein. METHODS: Recombinant hK6 protein was used as immunogen to develop polyclonal antibodies in rabbits and mice. These antibodies were used to develop a sandwich-type time-resolved immunofluorometric procedure for hK6. RESULTS: The newly developed hK6 immunofluorometric assay has a detection limit of 0.5 microg/L and upper concentration range of 200 microg/L. The assay is highly specific (no detectable cross-reactivity from PSA and hK2) and was used to quantify hK6 protein in various biologic fluids. Highest concentrations of hK6 were found in milk of lactating women, cerebral spinal fluid, nipple aspirate fluid, and breast cyst fluid. hK6 was also detected in male and female serum, in the majority of seminal plasmas and in a small fraction of amniotic fluids and breast tumor cytosols. hK6 was not detectable in urine. Chromatographic studies indicated that hK6 is present in these biologic fluids in its free, 30-kDa form. CONCLUSIONS: This is the first reported sensitive immunofluorometric procedure for quantifying hK6 protein. hK6 is a secreted proteolytic enzyme that is found at high levels in cerebrospinal fluid and all breast secretions. This assay will facilitate further studies to examine the possible application of hK6 in diagnostics, including cancer and neurodegenerative disorders.

Estrone and estradiol metabolism in vivo in human breast cysts.

Fibrocystic disease of the breast manifesting palpable cysts express breast cyst fluids frequently containing estrogen sulfates at concentrations far exceeding those found in sera of the patient. The study explored the potential of the breast cyst to synthesize some of these estrogen sulfates. Deuterated estrone and estradiol were synthesized and either (estradiol, 4 cases or estrone, 2 cases) was injected into a cyst. The cyst was aspirated at approximately 0, 4 and 8 h, the target being 1 ml, 50% and complete aspiration respectively. Metabolites were purified sequentially by ether extraction, enzymatic hydrolysis of estrogen conjugates, chromatography on Sephadex LH 20 and identified by gas chromatography linked to mass spectrometry. The unconjugated fraction isolated from the ether extract was subjected to the same purification and detection scheme. Among the conjugates, deuterated estrone sulfate was the major metabolite of either precursor in all studies, while estradiol sulfate was not detected in any of the 6 experiments. The sulfate fractions also yielded traces of 16alpha-hydroxyestrone (2 studies), 4-hydroxyestrone (4 studies) and 2-hydroxyestrone (1 study). In the unconjugated fraction, one study with deuterated estradiol, 4- hydroxyestrone was obtained. In one study with deuterated estrone, traces of 2-hydroxyestrone and 16alpha- hydroxyestrone were obtained. These novel data are significant because patients with fibrocystic disease are at slightly elevated risk for developing breast cancer and 16alpha-hydroxyestrone and 4- hydroxyestrone are reported carcinogens.

Esterase activity in human breast cyst fluid: associations with steroid sulfates and cations.

Human breast cyst fluid (BCF) contains an esterase that on the basis of electrophoretic mobility and response to inhibitors differs from those found in the plasma. From a total of 384 BCF samples analyzed for esterase using p-nitrophenyl hexanoate as substrate, 149 (39%) showed significant activity. The samples had been analyzed for the concentrations of the sulfates of estrone, estriol, dehydroepiandrosterone, as well as the potassium and sodium cations (K+/Na+). The data were submitted to statistical analysis using the Spearman rank order test. The esterase-positive samples exhibited a significant positive association with each of the steroid sulfates and the K+/Na+ ratios. Except for protein concentration, there was no significant correlation between the esterase-positive and esterase-negative cysts. These observations may have physiological significance in that high K+/Na+ ratio cysts have been related to the histological status of the cyst.