Potential hazards to embryo implantation: A human endometrial in vitro model to identify unwanted antigestagenic actions of chemicals.

Embryo implantation is a crucial step in human reproduction and depends on the timely development of a receptive endometrium. The human endometrium is unique among adult tissues due to its dynamic alterations during each menstrual cycle. It hosts the implantation process which is governed by progesterone, whereas 17ß-estradiol regulates the preceding proliferation of the endometrium. The receptors for both steroids are targets for drugs and endocrine disrupting chemicals. Chemicals with unwanted antigestagenic actions are potentially hazardous to embryo implantation since many pharmaceutical antiprogestins adversely affect endometrial receptivity. This risk can be addressed by human tissue-specific in vitro assays. As working basis we compiled data on chemicals interacting with the PR. In our experimental work, we developed a flexible in vitro model based on human endometrial Ishikawa cells. Effects of antiprogestin compounds on pre-selected target genes were characterized by sigmoidal concentration-response curves obtained by RT-qPCR. The estrogen sulfotransferase (SULT1E1) was identified as the most responsive target gene by microarray analysis. The agonistic effect of progesterone on SULT1E1 mRNA was concentration-dependently antagonized by RU486 (mifepristone) and ZK137316 and, with lower potency, by 4-nonylphenol, bisphenol A and apigenin. The negative control methyl acetoacetate showed no effect. The effects of progesterone and RU486 were confirmed on the protein level by Western blotting. We demonstrated proof of principle that our Ishikawa model is suitable to study quantitatively effects of antiprogestin-like chemicals on endometrial target genes in comparison to pharmaceutical reference compounds. This test is useful for hazard identification and may contribute to reduce animal studies.

In vitro-Ishikawa cell test for assessing tissue-specific chemical effects on human endometrium.

The human endometrium is a fertility-determining factor. Its receptivity during the implantation window may be altered by chemicals. Since human embryo implantation is unique chemical risk assessment cannot be based solely on animal studies. We established a tissue-specific in vitro test based on human endometrial adenocarcinoma (Ishikawa) cells. Progesterone receptor (PR) was selected as primary target gene for estrogenic effects. Changes of mRNA levels were investigated by reverse transcription quantitative real-time PCR. Sigmoidal dose-response curves for up-regulation of PR mRNA and EC(50) values were established for 17beta-estradiol, diethylstilbestrol and the weak xenoestrogen bisphenol A. Nonylphenol also had a clear PR mRNA up-regulating effect. Several other chemicals were characterized as negative compounds. Among them was methoxyacetic acid which may produce false positive results in reporter gene assays. Up-regulation of PR protein by 17beta-estradiol, diethylstilbestrol, bisphenol A and nonylphenol was confirmed by Western Blotting.

Expression patterns of CRH, CRH receptors, and CRH binding protein in human gestational tissue at term.

Recent research suggests a significant role for placental corticotropin-releasing hormone (CRH) in controlling human parturition. This paper describes the expression of CRH, CRH receptors 1 and 2, and CRH binding protein (CRH-BP) in gestational tissue in late pregnancy. Placenta, myometrium, decidua, and fetal membranes were collected after uncomplicated pregnancies at term caesarian section before the onset of labour. The localisation and mRNA expression of CRH, CRH receptors, and CRH-BP were studied by immunohistochemistry and reverse transcription (RT)-PCR. CRH receptors were detected in placenta, myometrium, decidua, and fetal membranes. We demonstrated for the first time the presence of CRH receptors on resident macrophages and on endothelial cells. CRH receptor 1 mRNA was detected in all tissues investigated by RT-PCR, whereas CRH receptor 2 mRNA was restricted to myometrium and decidua. CRH mRNA was widely expressed in all tissue under study. Novel findings are also presented on the expression of CRH-BP in the myometrium. This widespread expression of the CRH system in gestational tissue suggests a paracrine role for CRH in the birth process (e.g. effects on macrophages and endothelial cells).

Characterization and identification of cytochrome P450 metabolites of arachidonic acid released by human peritoneal macrophages obtained from the pouch of Douglas.

Cytochrome P450 metabolism of arachidonic acid (AA) was investigated in human peritoneal macrophages which play a central role in chronic pelvic diseases in women (for example in endometriosis). The formation of eicosanoids other than prostaglandins (PGs) by these cells is still unknown. In non-activated macrophages obtained from women in the reproductive age, the main [(3)H]-AA metabolites coeluted with epoxyeicosatrienoic acids, dihydroxyeicosatrienoic acids (DHETs) and hydroxyeicosatetraenoic acids (HETEs) in reverse-phase HPLC. After zymosan activation a shift to PGs pathway was observed. Treatment with low doses of 2,3,7,8-tetrachlorodibenzo- p -dioxin increased the formation of a metabolite coeluting with 5,6-DHET. By gas chromatography/mass spectrometry 5,6-DHET (after beta-naphthoflavone induction), and 14,15-DHET as well as 11,12-DHET (after AA stimulation) were identified as major epoxygenase metabolites, respectively. The enantioselective formation of 12(S)-HETE was demonstrated by chiral-phase HPLC. Our findings demonstrate that non-activated peritoneal macrophages produce substantial amounts of bioactive cytochrome P450 metabolites of AA.

Exposure of human endometrium to environmental estrogens, antiandrogens, and organochlorine compounds.

OBJECTIVE: To determine concentrations of environmental estrogens, antiandrogens, and organochlorine compounds in human endometrium and body fat. DESIGN: Cross-sectional, population-based study. SETTING: Patient recruitment was done at a university hospital; chemical analysis was performed in a specialized private laboratory. PATIENT(S): Premenopausal, unexposed women undergoing hysterectomy for uterine myoma. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Concentrations of environmental modulators in human endometrium and body fat were quantified by high-resolution gas chromatography/high-resolution mass spectrometry. RESULT(S): Among known endocrine modulators, the antiandrogenic p, p'-dichlorodiphenyl-dichloroethylene was found in the highest concentrations in endometrium (median 4.7 microg/kg wet weight) and body fat (median 446 microg/kg wet weight). Only trace amounts of the identified environmental estrogens beta-hexachlorocyclohexane, o, p'-dichlorodiphenyl-trichloroethane, bisphenol A, hydroxylated polychlorinated biphenyls, and genistein were found in the endometrium (median <1 microg/kg wet weight). As major organochlorine contaminants without endocrine activities, polychlorinated biphenyls and hexachlorobenzene were found. CONCLUSION(S): Our data demonstrate that nonchlorinated environmental estrogens do not build up cumulative tissue concentrations in the endometrium. The risk of reduced fertility because of ambient levels of environmental estrogens in the endometrium is negligible.

Formation of proinflammatory cytokines in human term myometrium is stimulated by lipopolysaccharide but not by corticotropin-releasing hormone.

Human term myometrium is poorly characterized as a source of proinflammatory mediators involved in parturition. We have investigated the basal expression of cytokines in myometrium, as well as the effects of CRH and lipopolysaccharide (LPS) on cytokine release. Explants from term myometrium were challenged with CRH or LPS (1 microg/mL each) in short-term tissue culture. Interleukin (IL)-1beta++, IL-6, IL-8, and tumor necrosis factor (TNF)alpha concentrations in the medium were quantified by enzyme immunoassay. The major cytokines released after 24 h were IL-6 and IL-8. All cytokines investigated were stimulated significantly by LPS (P: < 0. 05) but not by CRH. Messenger RNA levels of these cytokines were investigated by RT-PCR. IL-1beta+ and IL-6 messenger RNA were present in preterm and term myometrium before and during labor, whereas IL-8 and TNFalpha were expressed only by myometrium in active labor. Furthermore, myometrial CRH receptors and macrophages were characterized immunohistochemically. We conclude that human term myometrium is a site of production of proinflammatory cytokines and is involved in the inflammation-like reactions mediating the birth process. Cytokine release in term myometrium seems not to be under control of CRH.

Primary structure of maize chloroplast adenylate kinase.

This paper describes the sequence of adenylate kinase (Mg-ATP+AMP<-->Mg-ADP+ADP) from maize chloroplasts. This light-inducible enzyme is important for efficient CO2 fixation in the C4 cycle, by removing and recycling AMP produced in the reversible pyruvate phosphate dikinase reaction. The complete sequence was determined by analyzing peptides from cleavages with trypsin, AspN protease and CNBr and subcleavage of a major CNBr peptide with chymotrypsin. N-terminal Edman degradation and carboxypeptidase digestion established the terminal residues. Electrospray mass spectrometry confirmed the final sequence of 222 residues (M(r) = 24867) including one cysteine and one tryptophan. The sequence shows this enzyme to be a long-variant-type adenylate kinase, the nearest relatives being adenylate kinases from Enterobacteriaceae. Alignment of the sequence with the adenylate kinase from Escherichia coli reveals 44% identical residues. Since the E. coli structure has been published recently at 0.19-nm resolution with the inhibitor adenosine(5')pentaphospho(5')adenosine (Ap5A) [Müller, C. W. & Schulz, G. E. (1992) J. Mol. Biol. 224, 159-177], catalytically essential residues could be compared and were found to be mostly conserved. Surprisingly, in the nucleotide-binding Gly-rich loop Gly-Xaa-Pro-Gly-Xaa-Gly-Lys the middle Gly is replaced by Ala. This is, however, compensated by an Ile-->Val exchange in the nearest spatial neighborhood. A Thr-->Ala exchange explains the unusual tolerance of the enzyme for pyrimidine nucleotides in the acceptor site.

Adenylate kinase from plant tissues. Influence of ribonuclease on binding properties on Mono Q.

Adenylate kinases modulate the three adenine nucleotide pools and were found to be localized as isoenzymes in different tissues and organelles in animals and plants. For investigations of adenylate kinase isoenzymes from plant tissues different plant extracts were examined by anion-exchange chromatography. During investigations with the strong anion exchanger Mono Q, adenylate kinase activity eluted in the void volume. This void volume activity did not always occur, but depended on the age of the plants and light treatment. The nature of the factors affecting void volume activity could only be partially resolved. It could be shown that RNase treatment at the beginning of extraction led to the disappearance of void volume activity, whereas an untreated extract still showed this activity.