Wolfgang Schäfer

Arachidonate metabolism in human placenta, fetal membranes, decidua and myometrium: Lipoxygenase and cytochrome P450 metabolites as main products in HPLC profiles

Eicosanoids play a key role in pregnancy maintenance and parturition. We investigated the metabolism of arachidonic acid (AA) in short-term tissue cultures of placenta, fetal membranes, decidua and myometrium. Tissues were obtained from caesarean sections before the onset of labour after uncomplicated pregnancies. The released metabolites were analysed by high performance liquid chromatography (HPLC) and specific immunoassays. In radiotracer experiments tissues were labelled with [3H]-AA and metabolites released after incubation with calcium ionophore A23187 were profiled by HPLC. Decidua was more active in metabolizing AA (turnover 34 per cent) than myometrium (28 per cent), placenta (21 per cent) and fetal membranes (17 per cent). Main product in placenta, decidua and myometrium was 12-hydroxyeicosatetraeinoic (12-HETE) (decidua: 19 per cent of released radioactivity, myometrium 14 per cent, placenta 7 per cent). Fetal membranes formed 5-HETE as main product. Another major metabolite in placenta, fetal membranes and decidua was characterized by HPLC as 5(6)-epoxyeicosatrienoic acid. Only myometrium released appreciable amounts of prostaglandins in form of 6-keto-prostaglandin F1 alpha. In non-radioactive experiments formation of eicosanoids from endogenous AA was investigated by HPLC (fluorescence- and UV-detection) and immunoassays. These experiments confirmed the high production of 12-HETE and the low formation of prostaglandins. Our results suggest that the biological role of AA-metabolites, other than prostaglandins, have as yet been underestimated.

Eicosanoid production by intrauterine tissues before and after labor in short-term tissue culture

Prostanoid production by intrauterine tissues from pregnant and non-pregnant women has been studied intensively over the last decade. Little is known about the lipoxygenase metabolites of arachidonic acid (AA). The production of prostaglandins and HETEs by pregnancy specific human tissues was investigated in a short-term culture system. Tissue samples were obtained after uncomplicated pregnancies from placenta, fetal membranes and decidua of deliveries before (n = 6) and after the onset of labor (n = 8) and incubated for 1 hour in oxygenated HBSS. In the supernatant, PGE2, PGF2 alpha, 6-keto-PGF1 alpha and TXB2 were measured with RIA and 15-, 12- and 5-HETE with HPLC and UV-detection. The main AA-metabolite in all tissue incubations was 12-HETE. Decidua produced 12 to 28 times more prostaglandins than placenta and fetal membranes with 6-keto-PGF1 alpha as the main metabolite. The main cyclooxygenase derivative measured from placenta and fetal membrane incubations was TXB2. After labor, fetal membranes showed an increase in total prostaglandin (significant for PGE2) and a decrease in HETE synthesis. The physiologic significance of 12-HETE in reproduction is still poorly understood, but a shift in AA metabolism from HETEs to prostaglandins may be involved in the initiation of labor. Furthermore, these results point to different roles of the tissue compartments within the pregnant uterus for the parturition process.

Critical evaluation of human endometrial explants as an ex vivo model system: a molecular approach

The human endometrium is unique among adult tissues. Its functions are modulated by numerous hormones and mediators. The aim of this study was to evaluate the suitability of human endometrial explants for studying functional effects of chemicals and drugs on gene expression biomarkers. Endometrial tissues were obtained by aspiration curettage and cultivated for up to 24 h. Relative mRNA concentrations were determined by reverse transcription quantitative real-time PCR. Viability was assessed by light microscopy, lactate dehydrogenase assay and scanning electron microscopy. It was acceptable after 6 h of culture but reduced after 24 h. Culture-induced alterations of mRNA levels were found for progesterone receptor, estrogen receptor(α), leukemia inhibitory factor and cyclooxygenase-2 in tissues from all cycle stages. The suitability of the model to detect chemical effects was demonstrated by the down-regulation of cyclooxygenase-2 mRNA by chlormadinone acetate in proliferative and secretory endometrium. The model is mainly restricted by interindividual variations and varying tissue quality. An advantage is the preservation of tissue composition. We conclude that human endometrial explants are a complex model due to limited viability, difficult standardization and intrinsic alterations during culture. Experiments with this model should be performed over a limited time period under strictly controlled conditions.

Urinary excretion of 6-keto-PGF1α TXB2 and PGE2 in a rat animal model for preeclampsia-like syndrome

The etiology of pregnancy induced hypertension (PIH) is still unknown. The pathophysiology must be clarified. In this paper we present an animal model where hypertension in pregnant and non-pregnant rats was induced by an experimental reduction of uteroplacental blood flow. Thus, a preeclampsia-like syndrome could be studied under defined conditions. The eicosanoid system was investigated for pathophysiological alterations of the kidney by measuring urinary excretion of 6-keto-PGF1 alpha, TxB2 and PGE2 with radioimmunoassay at day 18 of pregnancy. First, in gravid control animals concentrations of all three prostaglandins were significantly elevated compared to non-gravid controls. However, in hypertensive gravid rats urinary concentrations of these prostaglandins fell even below the levels of non-gravid controls. The observed decrease was more pronounced for the vasodilatory 6-keto-PGF1 alpha and PGE2 than for the vasoconstrictive TxB2. Our results demonstrate that an experimental reduction of uteroplacental blood flow in the rat culminates in symptoms which clinically (hypertension, proteinuria) and pathophysiologically (eicosanoid system) resemble to preeclampsia.

Alterations of intrauterine eicosanoid production in pregnancy-induced hypertension: Decreased production of 12-hydroxyeicosatetraenoic acid in the placenta

The important role of eicosanoids in pregnancy-induced hypertension is generally accepted. Because of the lack of innervation of the uteroplacental vessels, humoral vasoactive factors are important for the regulation of vascular tone. Until now, mainly the balance of vasodilatative and vasoconstrictive prostaglandins has been studied. We were able to confirm their intrauterine imbalance in hypertensive pregnancies. In addition, the placental production of less known lipoxygenase metabolites has been analyzed in this study. Intrauterine tissues (30-100mg wet weight) were examined for their release of eicosanoids. Short term tissue cultures were performed in Hanks balanced salts solution (HBSS) at 37 degrees C in an atmosphere of 95% air/5% CO2 with and without incorporation of tritiated arachidonic acid. The arachidonate metabolites in culture media were analyzed by High Performance Liquid Chromatography (HPLC) with radioactivity detection or by enzyme immunoassays or radioimmunoassays, respectively. All intrauterine tissues released more lipoxygenase metabolites than cyclooxygenase metabolites with 12-hydroxy-eicosatetraenic acid (12-HETE) as their main metabolite. The placental release of 12-HETE was significantly decreased in hypertensive pregnancies. In hypertensive pregnancies the ratio TXB2/6-keto-PGF1 alpha synthesis was increased. Lipoxygenase metabolites, especially 12-HETE, seem to have important physiological and pathophysiological functions in the intrauterine compartment. Their biological role in this context needs further investigation.

Urinary excretion of 6-keto-PGF1α, TXB2, and PGE2 in pregnancy-induced hypertension and preeclampsia

Objective: Our intention was to investigate whether, in pregnancy-induced hypertension (PIH), preeclampsia is accompanied by an altered urinary excretion of 6-keto-PGF1α (a metabolite of prostacyclin PGI2), TxB2 (a metabolite of thromboxane A2) and PGE2.Methods: Our study included 59 PIH patients with a blood pressure > 140/90 mm Hg. Of them, 18 displayed a proteinuria > 300 mg/L and were classified as preeclamptic. As controls, 53 normotensive pregnancies were investigated. Urine samples were purified by solid-phase extraction and reversed-phase HPLC, and 6-keto-PGF1α TxB2, and PGE2 were quantified by radioimmunoassays.Results: Urinary excretion of vasodilatory prostaglandins in the third trimester was significantly reduced in hypertensive pregnancies compared to controls. Further reductions were observed in preeclamptic patients. Mean values > SEM are: 6-keto-PGFα: controls 926 ± 49 pg/mg creatinine (crea), PIH 694 ± 49 pg/mg crea, 0.01 < P < 0.001, preeclampsia 424 ± 65 pg/mg crea, P < 0.001; PGE2: con...

Chlormadinone acetate suppresses prostaglandin biosynthesis in human endometrial explants

OBJECTIVE To elucidate the mode of action of chlormadinone acetate (CMA) in reducing dysmenorrheic pain by studying the effects of CMA and dexamethasone (DEX) on messenger RNA (mRNA) abundance of cyclo-oxygenase-2 (COX-2), annexin-1 (ANXA1), glucocorticoid receptor (GR), progesterone receptor (PR), and concentrations of prostaglandin F(2α) (PGF(2α)) and leukotrienes B(4) (LTB(4)) and C(4) (LTC(4)) in human endometrial explants. DESIGN Ex vivo study. SETTING University hospital. PATIENT(S) Fifteen premenopausal patients undergoing surgery for benign gynecologic disorders. INTERVENTION(S) Endometrial explants were obtained by aspiration curettage and stimulated ex vivo with interleukin-1β before exposure to CMA or DEX; mRNA levels were determined via reverse transcription-quantitative real-time polymerase chain reaction, and concentrations of arachidonic acid metabolites by enzyme immunoassays. MAIN OUTCOME MEASURE(S) Messenger RNA levels of COX-2, ANXA1, PR, and GR; concentrations of PGF(2α), LTB(4), and LTC(4) in endometrial explants treated with CMA or DEX. RESULT(S) In IL-1β-treated explants COX-2 mRNA and PGF(2α), concentrations were significantly down-regulated by CMA but not by DEX. Chlormadinone acetate did not affect mRNA abundance of ANXA1, PR, and GR. CONCLUSION(S) Our data suggest that CMA is a suppressor of COX-2 expression. Comparison with DEX revealed that progestin-specific activity of CMA may mainly be responsible for suppression of prostaglandin biosynthesis in human endometrium.

Organochlorine compounds and xenoestrogens in human endometrium.

Numerous in vitro-studies have demonstrated weak estrogenic effects of a large number of environmental chemicals (Jobling et al., 1995; Soto et al., 1994; Colborn and Clement, 1992). A major limitation of in vitro-testing is that it does not account for pharmacokinetic properties of the test compounds. Many xenoestrogens contain hydroxy groups or ester bonds which render them to rapid biotransformation and elimination. However, due to chronic intake buildung up of equilibrium concentrations of those compounds cannot be excluded. Therefore, human exposure data of reproductive tissues with environmental chemicals, e. g. xenoestrogens, are a prerequisite for prioritizing potential harzardous compounds for further risk assessment. In our study we have investigated tissue concentrations of organochlorines and xenoestrogens (e. g. DDT, PCB, HCB, HCH, endosulfane, alkylphenols, bisphenol A, phthalates and butylated hydroxyanisol) (Fig. 1) in human endometrium and in body fat.

Formation of Cytochrome P450 Metabolites of Arachidonic Acid by Human Placenta

Saturated and polyunsaturated fatty acids can be oxygenated by cytochrome P450. The P450 pathway of the arachidonic acid cascade has been etablished in many organs, tissues and cells e. g. the kidney, the cardiovascular system, the nervous system, the skin, the corneal epithelium and in the platelets (1). Arachidonic acid can be oxygenated by P450 in several ways. Epoxidations lead to the regio- and enantioselective formation of four epoxyeicosatrienoic acids (EET’s). These epoxides are enzymatically degraded by hydrolases to the corresponding vicinal dihydroxy compounds (DHET’s). In another important class of P450 reactions arachidonic acid is hydroxylated to various HETE’s. Up to now only few reports about the P450 pathway in intauterine tissues have been published. Only in one investigation (2) a HPLC-peak cochromatographing with 14,15-EET was further characterised by its enhancement after α-naphthoflavone-treatment and its inhibition by SKF525A.

Intrauterine Eicosanoid Production in Uncomplicated and Hypertensive Pregnancies: Alterations of Placental 12-Hydroxyeicosatetraenoic Acid

Complicating about 10% of all pregnancies pregnancy-induced hypertensive diseases account for an enormous number of maternal deaths. At present, termination of pregnancy represents the only treatment of the cause. The important role of eicosanoids in pregnancy-induced hypertension is generally accepted1–2. As humoral vasoactive factors they are important for vascular tone regulation of the non-innervated uteroplacental vessels. In PIH and related diseases, these vessels fail to dilate normally, leading to a chronic preplacental decrease in circulation. A systemic lack of prostacyclin may also promote an increased vasoconstriction and coagulation disturbances in PIH leading to endothelial cell disorder, hemolysis and the vicious circle of disseminated intravasai coagulation3–4.