Volker Hanf

Input/output balance of estrogenic active compounds in a major municipal sewage plant in Germany

24 h samples of untreated and treated wastewater were taken in parallel from a modern municipal sewage plant in southern Germany in March and June 1998. After solid phase extraction, total estrogenic activity was quantitatively measured with a miniaturized E-screen assay and the levels of nine estrogenic phenolic chemicals analyzed by HRGC/LRMS. 17Beta-estradiol equivalent concentrations (EEQ) were 58 and 70 ng/l in the influent and 6 ng/l in the effluent, indicating that the load of estrogenic activity of the wastewater was reduced by about 90% in the sewage plant. Less than 3% of the estrogenic activity was found in the sludge. 4-t-octylphenol, 4-nonylphenol, bisphenol A, 2-hydroxybiphenyl, and 4-chloro-3-methylphenol were detected in the untreated wastewater at levels from 0.13 to 3.6 microg/l. 4-t-octylphenol, 4-nonylphenol, and bisphenol A were present in the effluent at concentrations from 0.16 to 0.36 microg/l, 2-hydroxybiphenyl and 4-chloro-3-methylphenol were not detectable. The contribution of the quantified levels of phenolic xenoestrogens to total estrogenic activity in the sewage was 0.7-4.3%.

Development of a sensitive E-screen assay for quantitative analysis of estrogenic activity in municipal sewage plant effluents

A simplified proliferation test with human estrogen receptor-positive MCF-7 breast cancer cells (E-screen assay) was optimized and validated for the sensitive quantitative determination of total estrogenic activity in effluent samples from municipal sewage plants. After solid phase extraction of 1 l sewage on either 0.2 g polystyrene copolymer (ENV+) or 1 g RP-C18 material and removal of the solvent, analysis of the extracts in the E-screen assay could be performed without any clean-up step. This was even possible with untreated sewage. Parallel extraction of four sewage samples on both different solid phase materials gave comparable quantitative results in the E-screen. A blank sample did not induce cell proliferation. As additive behaviour of the estrogenic response of single compounds was proven for two different mixtures each containing three xenoestrogens, total estrogenic activity in the sewage samples, expressed as 17 beta-estradiol equivalent concentration (EEQ), could be calculated comparing the EC50 values of the samples with those of the positive control 17 beta-estradiol. The detection limit of the E-screen method was 0.05 pmol EEQ/l (0.014 ng EEQ/l), the limit of quantification 0.25-0.5 pmol EEQ/l (0.07-0.14 ng EEQ/l). In total, extracts of nine effluent and one influent sample from five different municipal sewage plants in South Germany were analyzed in the E-screen. All samples strongly induced cell proliferation in a dose-dependent manner which was completely inhibited by coincubation with 5 nM of the estrogen receptor-antagonist ICI 182,780. The proliferative effect relative to the positive control 17 beta-estradiol (RPE) was between 30 and 101%. 17 beta-Estradiol equivalent concentrations were between 2.5 and 25 ng/l indicating a significant input of estrogenic substances via sewage treatment plants into rivers.

Validation and application of a rapid in vitro assay for assessing the estrogenic potency of halogenated phenolic chemicals

The E-Screen assay serves as an in vitro tool for the detection of estrogenic activity of chemicals and extracts of environmental samples. Based on the induction of proliferation in human estrogen receptor-positive MCF-7 breast cancer cells we could substantially simplify the assay. As one important step of validation we applied the modified assay for testing nine known xenoestrogens. We could confirm the results of other groups assuring the reproducibility of the E-Screen assay. The results provide evidence that the E-Screen assay is suitable for determination of estradiol equivalency factors (EEFs) for environmental estrogens to rank their estrogenic potency relative to the natural estrogen 17 beta-estradiol. Further, we used the optimized proliferation test to screen nine halogenated phenolic compounds for their possible estrogenic potency. Three widely applied chemicals expressed a weak receptor-mediated estrogenic activity: the flame retardant Tetrabromo-Bisphenol-A, the disinfectant 4-chloro-3-methylphenol, and the herbicide educt 4-chloro-2-methylphenol. Their estrogenic potencies were five to six orders of magnitude lower than that of 17 beta-estradiol.

Bilateral tubal pregnancy with intra-uterine gestation after IVF-ET : therapy by bilateral laparoscopic salpingectomy : a case report

A patient with heterotopic (combined) pregnancies after IVF-ET was treated by bilateral laparoscopic salpingectomy. The patient was delivered of a healthy newborn in the 38th week of pregnancy.

Influence of various dioxins on in vitro motility of human sperm

Abstract Human ejaculate was subjected to a 2 step Percoll® gradient fractionation to obtain highly motile spermatozoa. RPMI 1640 medium supplemented with 10% pooled human serum was contaminated with various defined congeners of dioxins to their respective solubility levels. Dioxin concentrations in the stock solutions ranged from 0.72 ng/ml (2,3,7,8-TCDD) to 6.09 ng/ml (1,2,3,4,6,7,8,9-OCDD). Sperm suspension in Hams F-10 medium was added to equal volumes of dioxin contaminated RPMI 1640 medium stock in glass tubes and incubated at 37°C under 5% CO2. Over a period of 60 hours the experimental and control tubes were sampled for motility measurements using a Hamilton Thorn HTM2030 automated motility analyzer at regular intervals. Results were plotted as means of triplicate readings +/− standard deviation. Control spermatozoa and treated cells did not noticeably differ in their respective loss of motility with time. Achievable dioxin concentrations in contaminated media, although clearly above levels found in body fluids of the average population, did not seem to impair motility in mature sperm after ejaculation.

Serotonin induces progesterone release from human granulosa cells in a superfused granulosa cell system

SummaryThe dynamics of the release of progesterone (P) by human granulosa cells (GCs) was investigated in a granulosa cell-superfusion system using serotonin (5-HT), propanolol (PR), or mianserin (MI). Granulosa cells were obtained from 8 women undergoing in-vitro fertilization and embryo transfer. Progesterone was measured using radioimmunoassay. Serotonin (100 pMol) induced P release from human GCs. This effect was specifically blocked by 100 pMol MI whilst 100 pMol PR was ineffective in this condition. The results suggest, that the superfused GC system is suitable for studying the dynamics of steroid secretion by human GCs.

Spontaneous and gonadotropin-releasing hormone induced human chorionic gonadotropin release from superfused JAR choriocarcinoma cells

SummaryJAR choriocarcinoma cells have retained several characteristics of normal trophoblasts and have been used as an in vitro trophoblast model. The superfusion system is suitable for the study of hormone synthesis and/or secretion. JAR cells were cultured and transferred to the superfusion system in order to evaluate the spontaneous hCG secretion and the effect of GnRH. The spontaneous hCG release showed a periodic pattern with a 48 min phase interval. In our system single cells and cell-clusters were superfused and there is a possibility that cell to cell connections might have an influence on the regulation of hormone synthesis and/or secretion. GnRH in 4×10−7 M and 4×10−6 M concentrations or 100 mM KCl caused an immediate hCG release from the JAR cells Repeated administration of GnRH resulted in a delayed hCG release which is probably related to the relatively small amount of hCG available from the storage granules or to the phase of spontaneous secretion.

Mammakarzinom und Umweltfaktoren

In den westlichen Industrielandern ist das Mammakarzinom der haufigste bosartige Tumor der Frau. Von besonderer Bedeutung ist der seit vielen Jahren in den meisten westlichen Industrienationen verzeichnete Inzidenzanstieg, der in den USA seit den 1940er Jahren im Schnitt 1% pro Jahr betrug und in Deutschland Mitte der 1990er Jahre erstmalig rucklaufig war. Uber den weiteren Verlauf des langfristigen Trends wird erst die nachste Schatzung Klarheit bringen. Ein Teil der Inzidenzsteigerung ist sicherlich auf die Veranderung des Reproduktionsverhaltens und die damit verlangerte Ostrogeneinwirkung auf das undifferenzierte Brustdrusenparenchym zuruckzufuhren. Dennoch kann der Anstieg, wie auch die hohe Inzidenz selbst, nur zu etwa 25-30% mit den heute bekannten Risikofaktoren erklart werden. Daher ist es von besonderer Bedeutung, sich mit den exogenen (Umwelt-) Faktoren auseinander zu setzen, die im Verdacht stehen, eine Rolle bei der Entstehung und Promotion des Brustkrebses zu spielen. Daruber hinaus sind Fragen nach der Ursache von Brusterkrankungen in der Praxis haufig. Die Befahigung, hier Rat geben zu konnen, wird von den Patientinnen als Kompetenzzuwachs erlebt.

Platelets in pregnancy induced hypertension

have used the graph over the years would ever go back to the blind guesswork of pre-SF days. One of the commonest objections to using the graph is that it will increase interference and the caesarean section rate. Our section rate for many years was 5% and has never gone above lo%! But the graph has many other uses; the early diagnosis of twins, the detection of polyhydramnios, the usefulness in estimating fatal size in breech and cephalo-pelvic disproportion and the indication of fetal abnormality as in anencephaly when S-F height measurement and dates seem to be inconsistent. Also, the estimation of gestation when dates are not known or are clearly wrong, these problems are our daily bread and butter. What seems most to deter Western obstetricians from using the SF graph is the ready availability of high-tech methods for assessing fetal well-being. It must be remembered that most of the world’s women are not looked after by obstetric teams with all the latest aids to hand, but are, if they are lucky, looked after by general doctors and midwives, who may have little or no interest or experience in obstetrics. If these doctors come out to Africa thinking that without high-tech machines there is nothing they can do to assess pregnant women, how can we ever reduce the appalling statistics? They need every assistance in making the best of a bad job and for experts to argue about what are significant statistics while babies are dying in thousands is contributing nothing to solving Third World problems. Western centres of excellence have as great a responsibility to mothers in the Third World as they have to their own patients. Failure to use the partogram is another example. The partogram is still not being used everywhere in the Third World because of the ‘we can take it or leave it’ attitude of obstetricians in the West. The Third World will only follow if they lead. What you have to do is imagine yourself in a rural centre with only your eyes, ears and hands to help you and see what you make of your patient’s condition then! We desperately need you to give the lead. Please don’t let us down. Ian Kennedy Bamalete Lutheran Hospital Box 6 Ramotswa Botswana

Loss of PAF-like activity from human embryo conditioned media (ECM) following HPLC separation

Recently a platelet activating factor (PAF)-like activity has been found in embryo conditioned media (ECM) and consequentially been termed embryo-derived PAF (EPAF). Yet it remains unclear whether the embryo-released molecule is in fact PAF or a PAF precursor or inductor in vivo. In this study we shall show that ECM did not induce platelet aggregation in vitro; however, it was possible to detect PAF-activity using the sensitive splenectomized mouse bioassay (SMB). Following lipid extraction, PAF activity was diminished, and after additional HPLC separation completely lost. We propose that the active fraction of ECM is lipid in nature but that this molecule is not PAF. We would rather suggest that this molecule induces the production of PAF by other cell types in vivo.