[Endocrine disruptors: hormone-active chemicals from the environment: a risk to humans?]. / Endokrin aktive Substanzen in der Umwelt (Endocrine Disruptors): Gefahr für den Menschen?

Many substances from the technical and natural environment can cause damage to the endocrine system. Animal tests show that so-called endocrine disruptors (ED), such as pesticides, fungicides, plasticizers (phthalates), bisphenol A (BPA), and organotin compounds can interfere with the endocrine system. In humans, it is difficult to attribute such changes to specific ED. Nevertheless, in vitro studies with human cells and tissues clearly show that ED are able to interfere with endogenous hormones, i. e. affecting the steroid hormone metabolism and intracellular signaling. Several clinical studies show that humans are also affected, including reproductive disorders like reduction of spermatogenesis, decreased testosterone production or malformation of the genitals or induction of tumors like mammary carcinoma. Facing the body of reports documenting the effects of ED, the European Union supported--inter alia--COMPRENDO, a project addressing risk assessment of particular ED in human and wildlife species, while the FDA supports the industry's actions to stop producing BPA-containing baby bottles and infant feeding cups. Some ED show an u-shaped dose response curve and specific ED have effects at levels dramatically lower than thought relevant to traditional toxicology, a phenomenon termed "Low Dose Impact". Further research is needed to clarify whether the observed findings represent associations or causal results.

Impact of androgenic/antiandrogenic compounds (AAC) on human sex steroid metabolizing key enzymes.

Various pesticides, industrial pollutants and synthetic compounds, to which human populations are exposed, are known or suspected to interfere with endogenous sex hormone functions. Such interference potentially affect the development and expression of the male and female reproductive system or both. Chemicals in this class are thus referred to as endocrine disruptors (ED). This emphazises on the relevance of screening ED for a wide range of sex hormone-mimicking effects. These compounds are believed to exert influence on hormonal actions predominantly by (i) interfering with endogenous steroids in that they functionally interact with plasma membrane-located receptors as well as with nuclear receptors both for estrogens and androgens or (ii) affecting the levels of sex hormones as a result of their impact on steroid metabolizing key enzymes. Essential sex hormone-related enzymes within the endocrine system of humans are aromatase, 5alpha-reductase 2 as well as specific sulfotransferases and sulfatases (so-called phase I and phase II enzymes, respectively). Using suitable human tissues and human cancer cell lines (placenta, prostate, liver and JEG-3, lymph node carcinoma of prostate (LnCaP) cells) we investigated the impact of 10 widely used chemicals suspected of acting as ED with androgenic or antiandrogenic activity (so-called AAC) on the activity of these sex hormone metabolizing key enzymes in humans. In addition, the respective effects of six substances were also studied as positive controls due to their well-known specific hormonal agonistic/antagonistic activities. The aim of this report and subsequent investigations is to improve human health risk assessment for AAC and other ED.

Transfection of dendritic cells (DCs) with the CIITA gene: increase in immunostimulatory activity of DCs.

Dendritic cells (DCs) are the major antigen-presenting cells. They are able to present tumor antigens to immunologic effector cells. MHC class II molecules on DC surfaces play an important role in priming effector cells against tumor cells and their antigens. The transactivator CIITA (MHC class II transactivator) is a non-DNA-binding transactivator, which regulates the expression of MHC class II, HLA-DM, and invariant chain and behaves as a master controller of constitutive and inducible MHC class II gene activation. Here, we transfected DCs with the CIITA gene using a novel transfection technique. The vector system consisted of a plasmid bound to an adenovirus via poly-L-lysine, which is covalently bound to a UV-irradiated adenovirus. After transfection, expression of MHC class II on DCs increased from 27% to 75% on day 2 after transfection. Transfected DCs were co-cultured with immunologic effector cells. Cytotoxicity of effector cells against tumor cells increased after co-culture with transfected DCs to 63% compared to 15% with effector cells co-cultured with irrelevantly transfected DCs (P=.037). This effect was dependent on the timing and period of co-culture. In conclusion, transfection of DCs led to an increase in antitumoral immunostimulatory capacity of DCs. We can further conclude that DCs could be efficiently transfected with the CIITA gene. Transfection of DCs led to an increase in antitumoral immunostimulatory capacity of DCs and may have a major impact on immunotherapeutic protocols for patients with cancer.

Significance of the CAG repeat length in the androgen receptor gene (AR) for the transactivation function of an M780I mutant AR.

Mutations in the androgen receptor gene (AR) cause a wide spectrum of androgen insensitivity syndromes (AIS). Mutation analysis of patients with AIS has revealed that the same missense mutation of the AR gene can give rise to strongly divergent phenotypes suggesting the influence of modifying factors. The polymorphic CAG repeat in the first exon of the AR gene may be such a modifying factor. The influence of the length of the CAG repeat on the transactivation function of the M780I-mutant AR (causing partial and complete AIS) has been determined by cotransfection of HeLa cells with various CAG-AR expression vectors and a highly androgen-responsive luciferase reporter gene construct. The transcriptional activity of the M780I mutant AR can be, in contrast to the wild-type AR, considerably enhanced by non-physiologically high androgen concentrations. Furthermore, an inverse relationship between the number of the CAG repeats in the mutant AR and its activity has been observed.

Glucocorticoid-recognizing and -effector sites in rat liver plasma membrane. Kinetics of corticosterone uptake by isolated membrane vesicles. III. Specificity and stereospecificity.

In previous papers we provided evidence for a glucocorticoid (GC) responsive site in a highly purified rat liver plasma membrane (PM) fraction, which has proved to be osmotically active, 'right side-out' vesicles, free of CBG, glucocorticoid receptors (GR) and ATP (J. Steroid Biochem. Molec. Biol. 42 (1992) 737-756 and 757-771). This site, now called 'GC importer', mediates active transmembrane transport of corticosterone (B). Pronounced specificity, including stereo- and enantiomeric specificity, of ligand-GC importer interaction was demonstrated by competition assays using 54 different steroidal hormones and molecules. Important structural prerequisites for ligands with high specificity for the GC importer are plane C21-steroid hormones with 1-ene and/or 4-ene or 5alpha-reduced configuration, and/or OH-group(s) at C11beta>C17alpha>C21. Unexpectedly, other preferred ligands are C17alpha-ethynyl steroids like estrogens with an OH- or OCH3-group at C3 (EE2, mestranol) as well as progestins with C3-OH and 4-ene configuration (ethynodiol). C21-steroids with 11alpha-OH, 11-keto, 16alpha-CH3, 16beta-CH3, 16alpha-OH or 5beta-reduced configuration are low specificity ligands. The importer even displays different specificity for enantiomers (levonorgestrel>L-norgestrel). Altogether, the GC importer preferentially recognizes active GC and natural progestins which act as GC-antagonist (e.g. prednisolone>11beta-cortisol = B > or = progestins). Synthetic GC-agonists (e.g. dexamethasone, betamethasone, triamcinolone), most synthetic progestins, biologically inactive GC (e.g. 11alpha-cortisol, prednisone, cortisone, 11-dehydro-B), mineralocorticoids (aldosterone), natural estrogens (e.g. E1, E2, E3), DES and vitamin D3 derivatives do not interact with the GC importer. Osmotic shrinkage experiments revealed that interaction of high as well as low specificity ligands with the GC importer comprises reversible binding and transport through the PM. The ligand specificity profile of the GC importer and the GR exhibit pronounced differences, suggesting that both GC recognizing sites are different proteins. Performing immunoblotting, using specific mono- and polyclonal antibodies directed against the intracellular rat GR, of the PM pretreated with the membrane protein solubilizing detergent CHAPSO, we found that specific steroid binding to the PM site is not due to contamination with GR. Colchicine, daunorubicine, quinine, reserpine, verapamil and vinblastine, representatives of lipophilic xenobiotics which are known to be transported out of cells by the glycoprotein P170, did not compete with B for uptake into PM-vesicles, indicating that the GC importer is not a member of the ABC/mdr superfamily. The GC importer seems to be an additional link in the chain of steroid signal transduction and may be functionally involved in the action of natural GC-agonists and GC-antagonists.

Biochemical monitoring of fetal distress with serum-immunoreactive erythropoietin.

The ability of parameters like umbilical arterial pH and Apgar score to predict and/or to reflect fetal distress are limited. It is known that erythropoietin (EPO) increases when partial pressure of oxygen is insufficient for metabolic demand. Therefore we studied the levels of EPO in the cord blood of stressed neonates (n = 75). In addition, reference values for EPO were established in a group of healthy term infants (n = 54) (mean +/- SD: 20.02 +/- [mU/ml]) and in premature infants (n = 77) according to gestational age. A significant increase in EPO concentrations was found in the stressed group: 153.4 +/- 418.8 [mU/ml], p < 0.003 (n = 27) in acute stress; and 102.6 +/- 127.1 [mU/ml], p < 0.002 (n = 48) in chronic stress. However parameters like hemoglobin, hematocrit, umbilical arterial pH and Apgar-score did not correlate with EPO values. A sensitivity of 59% and a specificity of 92% was calculated. We conclude that serum EPO concentrations are capable of detecting acute and chronic stress and could be useful as a screening method. In part EPO concentrations also allow us to grade stress in pregnancies that are complicated by diseases like preeclampsia.

[Erythropoietin as a biochemical parameter for fetal hypoxia]. / Erythropoietin als biochemischer Parameter für fetale Hypoxie.

The ability of parameters like umbilical arterial pH and Apgar-score to predict and/or to reflect fetal distress is limited. It is known that erythropoietin (EPO) increases due to hypoxic stimulation. Therefore we studied the levels of EPO in the cord blood of stressed neonates (n = 75). In addition, reference values for EPO were established in a group of healthy term infants (n = 54) (mean +/- SD: 20.02 +/- 6.4; median 17.8; range 8.7-40.3 (mU/ml]) and in premature infants (n = 77) according to gestational age (median/range: < 30 weeks 11.0, 5.5-17.5; 30-32 weeks 18.1, 5.5-136; 33-34 weeks 17.7, 8.3-422.9; 35-37 weeks 17.3, 5.5-272 [mU/ml]). EPO concentrations significantly increased in the stressed group: in acute stress (n = 27): mean 153.4, range 6.5-641.7 [mU/ml], p < 0.003; and in chronic stress (n = 48): mean 102.6, range 12.4-544 [mU/ml], p < 0.002. However, parameters like hemoglobin, hematocrit, umbilical arterial pH and Apgar-score did not correlate with EPO values. A sensitivity of 59% and a specificity of 92% was calculated. We conclude that serum EPO concentrations are capable of detecting acute and chronic stress. In part EPO also allows to grade stress in pregnancies, which are complicated by diseases like preeclampsia.

Mutations of the androgen receptor coding sequence are infrequent in patients with isolated hypospadias.

Androgen receptor defects can cause severe hypospadias. To examine the possibility that androgen receptor defects are a common cause of such deficiencies, we have determined the coding sequence of the androgen receptor gene in nine patients with severe hypospadias. The analysis of the androgen receptor coding sequence predicts a normal amino acid sequence for the androgen receptor of eight of the nine patients, indicating that the observed defects in virilization are infrequently caused by mutations of the open-reading frame of the androgen receptor. These findings demonstrate the importance of family history and endocrine studies in identifying patients likely to harbor coding sequence mutations in the androgen receptor gene, and they serve to focus attention on other genes that may influence androgen action in this group of patients.

Xanthine derivatives: comparison between suppression of tumour necrosis factor-alpha production and inhibition of cAMP phosphodiesterase activity.

Several in vitro and in vivo studies have demonstrated suppression of tumour necrosis factor-alpha (TNF-alpha) synthesis by pentoxifylline. In the present study we compared the effect of pentoxifylline with that of five other xanthine derivatives. We addressed two questions. First, what is the relative potency of those chemically related compounds in suppressing the lipopolysaccharide (LPS)-induced production of TNF-alpha in human mononuclear cells? Second, does suppression of TNF-alpha production by these xanthine derivatives correlate with their capacity to inhibit 3',5'-cAMP phosphodiesterase (PDE) activity? The experimental drug A 80 2715 [1-(5-hydroxy-5-methylhexyl)-3-methyl-7-propylxanthine] was identified as the most potent agent with an IC50 (concentration exerting 50% suppression of LPS-induced TNF-alpha production) of 41 microM (mean of 13 individuals). The IC50 values of the other substances ranged between 106 microM for HWA 138 and 419 microM for theophylline. The LPS-induced interleukin-1 beta (IL-1 beta) production was not influenced by all substances tested at comparable concentrations. Inhibition of PDE activity was determined in a cell-free system using PDE isolated from bovine heart. All xanthine derivatives dose-dependently inhibited PDE activity. Furthermore, with the exception of theophylline, there was a high degree of correlation between the potency to suppress TNF-alpha production in the cell culture system and the potency to inhibit PDE activity in the cell-free enzymatic assay. This argues for a crucial role of PDE inhibition in the suppression of TNF-alpha synthesis by xanthine derivatives.

Glucocorticoid-recognizing and -effector sites in rat liver plasma membrane. Kinetics of corticosterone uptake by isolated membrane vesicles--II. Comparative influx and efflux.

To elucidate the initial step in the interaction between glucocorticoids (GC) and the hepatocyte, we examined at 22 degrees C further kinetic properties of active corticosterone (B) transport mediated by a putative, plasma membrane-inserted carrier for GC (GCC) as previously reported [Alléra and Wildt, J. Steroid Biochem. Molec. Biol. 42 (1992) 737-756]. We used a purified, well-characterized, osmotically active vesicle fraction of plasma membrane (PM), free of ATP, isolated from rat liver and a method developed by us to describe transport processes mathematically: (1) uptake (U) of 7 nM B into the vesicles (influx, I) occurred very rapidly whereby T1/2 = 8.3 s, the time (S) required for half maximum transport; the influx velocity (dU/dS = V) decreased degressively with time following second-order kinetics characterized by an initial transport V (VT0) of 177.7 fmol/mg membrane protein/s. (2) VToI of B-influx rose with temperature biphasically (P less than 0.025): activation energy above and below 15 degrees C (at PM phase transition) amounted to 9.5 and 26.5 kJ/mol. Neither at 45 nor at 60 degrees C did transport take place, revealing the high thermolability of GCC. (3) Efflux (E) of 6.5 nM B, i.e. transport out of the vesicles preincubated with the steroid, showed that influx had resulted in a 19.6-fold intravesicular hormone accumulation, indicating active ("uphill") transport. (4) The efflux velocity (dE/dS = V) exhibited almost the same kinetic quality as that of influx: it decreased following mainly second-order kinetics whereby T1/2 = 8.0 s. However, its whole time-course was much slower and the VT0 of efflux (VToE) was 6.3 lower than VToI. (5) Using physics and thermodynamics, we deduced that the affinity (AF) between B and GCC is proportional to the square of VT0. (6) Thus, because AF approximately (1/6.3)2, AF of the B-GCC interaction after completion of influx was calculated to be 40 times lower (Kd = 708 nM; delta G degrees = -34.9 kJ/mol) than at outset of influx, whereby delta G degrees = -44.0 kJ/mol. Concluding from these and previous findings, we present a new hypothesis on B transport into the hepatocyte: There is no difference (P greater than 0.3) in free enthalpy between transcortin (CBG) and the intracellular GC receptor interacting with B (delta G degrees = -40.1 and -40.4 kJ/mol). The GCC, however, is characterized by its ability to switch from a high- to lower-affinity when interacting with B (and vice versa due to metabolic energy input).(ABSTRACT TRUNCATED AT 400 WORDS)

Glucocorticoid-recognizing and -effector sites in rat liver plasma membrane. Kinetics of corticosterone uptake by isolated membrane vesicles--I. Binding and transport.

To gain insight into the mechanisms governing cellular uptake of glucocorticoids, we studied the binding and membrane transport of corticosterone (B) on a highly purified plasma membrane fraction from rat liver that was homogenized using a gentle, isotonic procedure. The fraction was mostly in the form of right-side out and osmotically active vesicles that were free of intracellular glucocorticoid receptors (GCR), transcortin (CBG) and ATP. Our uptake and binding studies carried out at 22 degrees C with [3H]B in physiological concentrations resulted in the following findings: (1) unlabeled B competed with [3H]B for uptake by the membrane vesicles; half-maximal competition of specific uptake was achieved with a 10- to 11-fold molar excess of unlabeled B. (2) [3H]B uptake was a saturable process of unusual kinetics (multiple sigmoidity); modified Scatchard plots revealed three significantly different apparent Kd-values of 1.3, 4.7 and 17.3 nM, corresponding to free B in the blood of non-stressed rats (4-16 nM). (3) Osmotic shrinkage of the vesicles led to a linear decrease in specific uptake, while non-specific uptake was independent of vesicle volume. Passive diffusion of [3H]B took place in leaky, but not in intact, vesicles. Reversible binding to, and mediated transport through, the membrane were interdependent parts of a strongly linked process. B was accumulated inside the vesicle up a concentration gradient by an active transport that followed first-order kinetics (Kt:3.9 nM); for its statistically reliable mathematical formulation and kinetic analysis, a replot was developed that revealed that relative accumulation increased with decreasing external hormone concentration. (4) Comparative binding studies disclosed that the apparent Kd-values (86.5 +/- 7.3 and 77.0 +/- 14.3 nM, respectively) of the [3H]B interactions with CBG and GCR did not differ (P greater than 0.3). These findings permit the conclusion that a plasma membrane-inserted carrier for B, effectively operating at physiological concentrations in the blood, is involved in a functional and regulatory manner in the biological action of glucocorticoids.

Characteristics and specificity of the glucocorticoid "carrier" of rat liver plasma membrane.

The uptake of corticosterone by highly purified plasma membrane vesicles of rat liver was studied by a rapid-centrifugation technique which allows uptake measurements within 5 s. The vesicles are free of soluble cytoplasmic constituents. Therefore, association of hormone with the vesicle is attributed entirely to components of the vesicle-membrane. Half maximal uptake is reached at 8 s at 21 degrees C. At 15 degrees C transition of the lipid state in the membrane leads to a decrease of uptake, a characteristic property common to membrane mediated processes. The uptake of corticosterone is saturable and reversible but does not follow normal saturation kinetics. The apparent dissociation constants of three uptake systems bear direct relation to the concentration of free corticosterone in rat plasma (4-16 nM) supporting a physiological role for the system. Uptake of corticosterone decreases with decreases in vesicular volume; about 50% of the hormone is bound specifically and 50% is transported to the lumen of the vesicle. Since outflow of intravesicular hormone also occurs readily, the uptake and transport is proposed to be mediated by putative "carriers". The "carrier" preferentially transports glucocorticoids; dexamethasone is not taken up by this putative molecule. Steroids with 5 alpha conformation are more potent inhibitors of the "carrier" for corticosterone than 5 beta-steroids. Androgens and estrogens are weak competitors of corticosterone. The affinity of the "carrier" for several hormones differs considerably from that of the cytoplasmic receptor. Morris hepatoma cells (MH 3924) do not take up corticosterone. Our results prompt us to propose the hypothesis that the transport function of the "carrier" and the binding of the hormone by the cytoplasmic receptor are two different entities; perturbation of the "carrier" may lead to steroid unresponsiveness. Normal expression of steroid hormone activity is manifested in the concerted action of the functionally sound cell membrane "carrier" and the intracellular receptor.