Biomedical subjects
Publications and source records attributed to L Markiewicz.
The goal of this paper was studying the effectiveness of frequently repeated low values of long duration acceleration on circulatory system response during gradual onset rate of acceleration on a human centrifuge.
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Estrogenic activities of testosterone (T) and 5a-dihydrotestosterone (DHT) were detected and measured by using their specific stimulatory effects on alkaline phosphatase (AP) activity in human endometrial adenocarcinoma cells of the Ishikawa Var-1 line. These two physiologic androgens were able to induce, at microM concentrations, estrogenic effect believed to be mediated by the estrogen receptor (ER) since the antiestrogens ICI-164384 and 4-hydroxytamoxifen (OHTam), but not the antiandrogens hydroxyflutamide (OHFl) or cyproterone acetate (CPA), reversed that effect. By using another in vitro bioassay, based on the progestin-specific stimulation of AP activity in cells of the T47D human breast cancer line, progestagenic activity was detected and measured in T, DHT and three synthetic androgens: nandrolone (19-nortestosterone). 7 alpha-methyl 19-nortestosterone (MENT) and mibolerone (7 alpha, 17 alpha-dimethyl 19-nortestosterone) (DMNT). While progestagenic effects of T and DHT required relatively high concentrations (microM levels), the synthetic androgens stimulated AP activity at nM or pM levels. These effects seem to be mediated by the progesterone receptor (PR), since they are completely abolished by the antiprogestins RU-486, ZK-98299 and ZK-112993, but not by the antiandrogen OHFl. These simple in vitro bioassays, expressing biological effects of the test compounds in human cells in culture, revealed dual or multiple hormonal activities coexisting in a single compound and provide quantitative information of considerable pharmacological importance concerning the complex actions of drugs.
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In the years 1986-1990 within Upper Silesia Region (Katowice Voivodeship) 1240 larynx cancer cases were diagnosed. Larynx cancer standardized incidence rates there are: 12,5 cases per 100 thousand on the whole area, maximal--28.6 and minimal--2.2 per 100 thousand, taking into consideration 93 administrative units (45 towns and 48 community councils). Larynx cancer incidence rates were dynamically increasing every year by 3%. There has not been attained geographic correlation between highest larynx cancer incidence level and highest ambient air pollution.
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The physiologic mechanisms whereby the human endometrium maintains hemostasis during endovascular trophoblast invasion, yet permits menstrual hemorrhage, are unknown. This paradoxical relationship was investigated by evaluating endometrial expression of tissue factor (TF), the primary initiator of hemostasis, and plasminogen activator inhibitor-1 (PAI-1), the primary inhibitor of fibrinolysis. We observed increased immunostaining for TF and PAI-1 in sections of decidualized stromal cells from luteal phase and gestational endometrium. To determine whether TF and PAI-1 expression are directly linked to decidualization, both endpoints were monitored in a well described in vitro model of decidualization. Thus, confluent stromal cell cultures were exposed to vehicle control, 10(-8) M estradiol (E2), 10(-8) to 10(-6) M medroxyprogesterone acetate (MPA) or both E2 + MPA for 2-24 days in serum-containing or defined media. The progestin enhanced the content of stromal cell-associated immunoreactive and functionally active TF and PAI-1 released into the medium and elevated levels of stromal cell TF and PAI-1 mRNA. While E2 alone was ineffective, it greatly augmented MPA-enhanced TF and PAI-1 protein and mRNA content. Dose-dependent effects on TF and PAI-1 content were observed between 10(-8) to 10(-6) M MPA +/- E2. Similar results were observed for decidual cells derived from first trimester endometrium and cultured in type 1 collagen gels. Following optimal induction of TF and PAI-1 expression by E2 + MPA in stromal cell cultures, removal of these steroids greatly reduced levels of both TF and PAI-1 protein and mRNA within 4 days. These studies suggest a mechanism whereby endometrial hemostasis is maintained during trophoblast invasion yet reduced at the end of nonfertile cycles to permit menses.
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The progestin-specific stimulation of alkaline phosphatase (AP) activity in cells of the T47D human breast cancer line was applied to the development of a sensitive microtiter plate bioassay for the quantitative evaluation of progestagenic and antiprogestagenic potencies of natural and synthetic compounds. Some of the steroids tested (viz. progesterone, medroxyprogesterone acetate, norethynodrel) behaved as full-agonists, capable of inducing AP activities to the same maximal levels (equal efficacy), while others (norethindrone, gestrinone, R5020, norgestrel, Org OD 14 and its 4-ene metabolite) behaved as partial agonists, eliciting lower maximal effects. Efficacy, EC50 values (concentrations at which they induce one-half of the maximal response) and "slope factors" serve to characterize agonistic effects. Relative progestagenic potencies among the full-agonists were evaluated by comparing EC50 concentrations. Several 19-nor synthetic progestins (norethynodrel, norethindrone, Org OD 14 and its 4-ene isomer, dl-norgestrel, levo-norgestrel, RU2323), but none of the tested progestins with the pregnane structure, showed intrinsic estrogenic activity, as evaluated by using a similar in vitro bioassay based on a previously reported estrogen-specific induction of AP in human endometrial adenocarcinoma cells of the Ishikawa Var-1 line. Maximal estrogenic effects of all the tested progestins with dual activity were as high as those of estradiol. However, these compounds widely varied in their EC50 values for estrogenic activity. Consequently, the in vitro bioassays can reveal differences in the ratio of progestagenic and estrogenic activities intrinsic to these compounds. The reduced capability of the partial agonists to exert progestagenic or estrogenic effects on AP expression may reflect an impeded, receptor-mediated action, a mechanism that would also account for their inhibitory effects on the induction of AP activity by full agonists. Partial progestagenic agonists were able to reduce the efficacy of a full agonist to their own partial maximal activity.
The extracellular matrix (ECM) protein fibronectin (FN) is a critical regulator of uterine-placental adherence. In the present report we compared the effects of glucocorticoids on FN expression in cytotrophoblast cultures isolated from human first trimester and term placentas to elucidate potential steroid-dependent cellular mechanisms associated with human parturition. Based on immunoassays, treatment of first trimester cytotrophoblasts with 10(-7) M dexamethasone (DEX) for 2 cr 4 days reduced medium levels of oncofetal FN (onfFN; i.e. FNs bearing an oncofetal epitope) to approximately 80% of control levels. Conversely, treatment of cytotrophoblasts isolated from term placentas with DEX dramatically reduced medium levels of onfFN to approximately 12% of control values. Treatment of both first trimester and term cells with 10(-6) M progestin, mineralocorticoid, or estrogen had no significant effect on onfFN expression in either cell type. Glucocorticoids specifically down-regulated medium levels of onfFN in term cells, but not in first trimester cells. In contrast, DEX treatment promoted an approximately 3- to 7-fold increase in levels of hCG in both first trimester and term cytotrophoblasts, suggesting that the effects of glucocorticoid on FN and hCG expression are elicited through independent cell-signaling pathways. In first trimester cells, DEX promoted a reduction in rates of FN and laminin synthesis to 60-70% of control levels. In term cells, DEX treatment reduced levels of FN and laminin synthesis to approximately 10% of control levels. Similarly, DEX treatment down-regulated levels of FN mRNA to approximately 60% and 10% of control values in first trimester and term cells, respectively. The first trimester of human pregnancy is associated with low levels of glucocorticoids and reduced glucocorticoid responsiveness. These conditions would favor high levels of placental ECM protein synthesis, thus stabilizing uterine-placental adherence. Conversely, elevated levels of glucocorticoids near parturition and increased glucocorticoid responsiveness would inhibit placental ECM protein synthesis, reducing uterine-placental adherence and promoting the separation of placenta from uterus.
In vitro conversion in human endometrial tissue of Org OD 14 [17 alpha-hydroxy-7 alpha-methyl-19-norpregn-5(10)-en-20-yn-3-one, a 3-keto-delta 5-10-19-nortestosterone derivative structurally related to norethynodrel] to its 4-ene isomer was demonstrated and measured spectrophotometrically and by chromatographic separation of the labeled metabolite from the tritiated precursor. The endometrial isomerase catalyzing this conversion is the 3 beta-hydroxy-steroid dehydrogenase/isomerase (3 beta HSD/isomerase), detected by Western blotting as a 42 kDa band, as confirmed by the inhibition of Org OD 14 isomerization with an antibody against this enzyme. The endometrial isomerase activity was found to be higher in secretory than in proliferative tissue and to be influenced by progestins, as suggested by the small but significant increase in activity resulting from exposure of proliferative endometrium to medroxyprogesterone acetate under organotypic culture conditions. In addition to the expected physiologic importance of endometrial 3 beta HSD/isomerase in the local metabolism of circulating steroids of adrenal origin, its presence in the endometrium is likely to have pharmacologic relevance, as illustrated by the local conversion of Org OD 14 to the 4-ene isomer, a metabolite with higher progestagenic and lower estrogenic potencies than those of its precursor. The local, tissue-specific, modification of the precursor would yield intracellular concentration ratios of Org OD 14 to 4-ene isomer in the endometrium significantly lower than those in blood. As a result, the estrogenic effects of Org OD 14 or of its 3-hydroxy metabolites on endometrial cell proliferation are minimized by the local formation of the progestagenic 4-ene isomer. This is a favorable feature of Org OD 14 since it selectively prevents undesirable proliferative stimulation of the endometrium in postmenopausal users while preserving its beneficial effects on other tissues, including bone.
Some of the isoflavonoids present in human diet as well as in urine are expected to exert biologic effects as they have been reported to bind to estrogen receptors and to be estrogenic in other species. This report describes the in vitro assessment of estrogenic effects of isoflavonoids using human endometrial cells and tissue. The relative estrogenic potencies (EC50 values) of estradiol, 3 dietary isoflavonoids (coumestrol, genistein and daidzein) and one of their metabolites (equol), were estimated by using a recently developed multiwell plate in vitro bioassay based on the estrogen-specific enhancement of alkaline phosphatase (AlkP) activity in human endometrial adenocarcinoma cells of the Ishikawa-Var I line. The maximal AlkP activity elicited by the isoflavonoids tested was as high as that achieved with estradiol and their effects were suppressed by the antiestrogens 4-hydroxytamoxifen and ICI 164,384. These results indicate that estradiol and the isoflavonoids exert their effects on AlkP by similar interactions with the estrogen receptor, with potencies depending on binding affinities. The estrogenic effect of equol was confirmed by another in vitro bioassay, based on the estrogen-stimulated enhancement of prostaglandin F2 alpha output by fragments of human secretory endometrium.
Oncofetal fibronectin (onfFN) is a uniquely glycosylated form of FN suggested to play a critical role in uterine/placental adherence during pregnancy. In the present study we have examined steroid regulation of onfFN in highly purified preparations (> or = 95%) of cytotrophoblasts isolated from human term placentas. Based on immunoassays, relative to controls, treatment of cytotrophoblasts with 10(-6) M medroxyprogesterone acetate (MPA) down-regulated media levels of onfFN 25, 53, 59, and 62% on days 1, 2, 3 and 4, respectively. The pattern of steroid regulation and levels of total FN were nearly identical to that of onfFN suggesting that chronic steroid treatment regulates synthesis of FN and not its oncofetal glycosylation. MPA treatment induced a 2-fold stimulation in media levels of hCG indicating that increased placental function was associated with steroid-mediated changes in FN expression. Steroid specificity experiments demonstrated that MPA, cortisol, and dexamethasone were potent inhibitors of onfFN expression whereas estradiol (E2), deoxycorticosterone, testosterone, progesterone, and the synthetic progestin OD-14, were not. This suggested that glucocorticoids and not progestins may be the physiologic regulators of placental FN expression and that MPA may mediate its matrix-modifying activity through a glucocorticoid-like mechanism. Treatment of cells with dexamethasone (10(-7) M) did not affect the levels of total protein synthesis or the release of human placental lactogen to the culture medium. This indicated that steroid-mediated down-regulation of onfFN expression in cytotrophoblasts did not result from a general reduction of protein synthesis. Based on densitometric scanning of Western blots, MPA and dexamethasone treatments down-regulated media levels of onfFN 70% relative to control levels. Northern blotting revealed that MPA and dexamethasone mediated a 60-90% reduction in steady state levels of FN mRNA in the presence or absence of E2. Our in vitro model may provide a unique system to evaluate steroidal effects on extracellular matrix (ECM) protein expression. In addition, we suggest that steroids may critically regulate placental ECM protein synthesis, and thus affect trophoblast/uterine adherence throughout pregnancy and expulsion of the placenta and membranes following delivery of the fetus.
This study examined steroidal regulation of tissue factor expression by cultured endometrial stromal cells. Confluent stromal cell cultures derived from cycling human endometria were exposed to vehicle control, 10(-8) mol/L estradiol (E2), 10(-8)-10(-6) mol/L medroxyprogesterone acetate (MPA), or both E2 and MPA for 2-24 days in serum-containing medium. The progestin enhanced immunoreactive and functionally active stromal cell tissue factor content, achieving peak effects by 8-12 days of culture. Although E2 alone was ineffective, it augmented MPA-enhanced tissue factor content by 8 days of culture, with continued increases beyond 20 days. Dose-dependent effects on tissue factor protein content were observed between 10(-8)-10(-6) mol/L MPA added alone or together with E2. The content of tissue factor mRNA was also increased by MPA and synergistically increased by E2 plus MPA. Similar steroidal effects on stromal cell tissue factor protein and mRNA content were observed using a defined medium. After optimal induction of tissue factor expression by E2 plus MPA, removal of these steroids reduced levels of stromal cell tissue factor mRNA and protein, with virtually complete reversal by day 7 of withdrawal. These time-course and dose-response relationships establish in vitro conditions with which to dissect factors controlling endometrial hemostasis, whereas the observed effects of steroid withdrawal establish a novel model for the study of mechanisms regulating normal and abnormal uterine bleeding.
The intrinsic estrogenic activity of some progestins cannot be properly evaluated by using hormone responsive systems when the chosen end-points are also sensitive to progestagenic activity, usually antagonistic of estrogenic actions. We have therefore applied to the evaluation of some drugs commonly used in contraceptive and hormone replacement formulations a recently developed in vitro method to estimate estrogenic activities, which is based on measurements of the estrogen-stimulated alkaline phosphatase activity in cells of the Ishikawa-Var I human endometrial adenocarcinoma line, a response not influenced by progestins. Whereas progesterone, medroxyprogesterone acetate and danazol were found to be devoid of estrogenic activity in this assay, Org OD-14, norethynodrel, gestrinone (R 2323), norethindrone and dl-norgestrel provoked half maximal increases in alkaline phosphatase activity at concentrations (EC-50) of 7, 14, 140, 200 and 2900 nM, respectively, under conditions in which the corresponding value for estradiol was 8 pM. This intrinsic estrogenic activity can be inhibited by antiestrogens, as verified by reversing the effect of R 2323 with 4-hydroxytamoxifen. Since prostaglandin F2 alpha output by secretory endometrium is increased by estrogens and diminished by progestins, this end-point can serve to evaluate the net effect of drugs with intrinsic estrogenic and progestagenic activities. For instance, R 2323 showed estrogenic activity in this assay whereas Org OD-14 did not. The same in vitro system can be used to evaluate estrogen antagonistic activities of test compounds, using estradiol as the agonist. These in vitro systems are useful in establishing a profile of activities of a drug on a relevant human target tissue, in the screening of synthetic or natural compounds under investigation, and in studies on structure/action relationships.
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The human endometrial model for in vitro evaluation of estrogenic, estrogen antagonistic, and progestagenic effects of endogenous steroids, natural products or synthetic drugs was applied to the study of Org OD-14, an analog of norethynodrel developed by Organon International, Oss, The Netherlands, and some of its metabolites. Estrogen antagonistic actions of Org OD-14 and its 4-ene isomer were evident from their ability to suppress the enhancement of PGF2 alpha output elicited by estradiol on fragments of secretory endometrium and to decrease the rate of output of the prostaglandin by proliferative tissue, already stimulated by endogenous estrogens. These inhibitory effects were similar to those obtained with progesterone and do not appear to involve competition for the estrogen receptor since the antiestrogen 4-hydroxyamoxifen was not active in parallel incubations of proliferative endometrium. The progestagenic effects of Org OD-14 and its 4-ene isomer were also evident from their capability to enhance estradiol 17 beta-dehydrogenase activity and glycogen accumulation in specimens of proliferative endometrium. Estrogenic effects of the 3 alpha- and 3 beta-hydroxy metabolites of Org OD-14 were demonstrated by their stimulatory actions on PGF2 alpha output during incubations of secretory endometrium. The estrogenic and progestagenic actions of these compounds are in general agreement with their relative affinity for binding to the estradiol and progesterone receptors, although their actions may be influenced by intracellular metabolism in the endometrial tissue. For instance, the similarity in progestagenic activity of Org OD-14 and the 4-ene isomer, contrasting with their different affinities for the progesterone receptor, may result from in situ isomerization of Org OD-14 to the 4-ene metabolite.
We have developed an estrogen bioassay using the Ishikawa human endometrial adenocarcinoma cell line growing in 96-well microtiter plates. Alkaline phosphatase enzyme activity (AlkP) in these cells is markedly stimulated by estrogens, and this enzyme can be easily quantified in situ using a chromogenic substrate. These cells are very sensitive to estrogens; estradiol induces AlkP at levels as low as 10(-12) M. Antiestrogens completely block the action of estradiol. Various estrogens stimulate AlkP with potencies comparable to those achieved in vivo. The induction of AlkP is specific for estrogens; no other type of steroid, including androgens, progestins, mineralocorticoids, or glucocorticoids produce this effect. The stimulation of AlkP in Ishikawa cells is specific for estrogens, is highly reproducible and sensitive, and permits large numbers of samples to be assayed with ease. We have used this assay to investigate the estrogenic action of the adrenal delta 5-3 beta-hydroxysteroids. While pregnenolone is inactive, dehydroepiandrosterone and its sulfate ester induce AlkP slightly. However, the C19 steroid, 5-androstene-3 beta, 17 beta-diol is considerably more estrogenic in this assay, although it stimulates Ishikawa AlkP with a potency of 1/30,000 that of estradiol. The stimulation by 5-androstene-3 beta,17 beta-diol is inhibited by antiestrogens, but it is not blocked by the delta 5-3 beta-hydroxysteroid isomerase/dehydrogenase inhibitor, cyanoketone, or by the aromatase inhibitor, 4-hydroxy-androstenedione. Thus, neither conversion to a delta 4-3-ketone nor aromatization is required for the action of this unusual estrogen.