A prospective whole-mixture approach to assess risk of the food and chemical exposome.

Many widely used chemicals result in ubiquitous human exposure from multiple sources, including diet. Legislation mainly deals with the toxicological evaluation of single substances owing to a methodological and conceptual lack of alternatives, and does so within defined silos subject to over 40 distinct regulations in the EU alone. Furthermore, much of the research and many of the initiatives concerned with the assessment and evaluation of chemical mixtures and their potential effects on human health rely on retrospective analysis. Here we propose an approach for the prospective identification, assessment and regulation of mixtures relevant to human health. We address two distinct aspects of toxicology-which chemicals actually do occur together, and how potential mixture-related health hazards can be predicted-with an adapted concept of the exposome and large-scale hazard screens. The proactive use of the likelihood of co-exposure, together with the new approach of methods-based testing, may be a timely and feasible way of identifying those substances and mixtures where hazards may have been overlooked and regulatory action is needed. Ideally, we would generate co-exposure patterns for specific consumer groups, depending on lifestyle and dietary habits, to assess the specific risk of identified mixtures.

Workshop on the validation and regulatory acceptance of innovative 3R approaches in regulatory toxicology - Evolution versus revolution.

At a joint workshop organized by RIVM and BfR, international experts from governmental institutes, regulatory agencies, industry, academia and animal welfare organizations discussed and provided recommendations for the development, validation and implementation of innovative 3R approaches in regulatory toxicology. In particular, an evolutionary improvement of our current approach of test method validation in the context of defined approaches or integrated testing strategies was discussed together with a revolutionary approach based on a comprehensive description of the physiological responses of the human body to chemical exposure and the subsequent definition of relevant and predictive in vitro, in chemico or in silico methods. A more comprehensive evaluation of biological relevance, scientific validity and regulatory purpose of new test methods and assessment strategies together with case studies that provide practical experience with new approaches were discussed as essential steps to build up the necessary confidence to facilitate regulatory acceptance.

Preeclampsia is associated with loss of neuronal nitric oxide synthase expression in vascular smooth muscle cells of the human umbilical cord.

AIMS: Umbilical blood vessels are not innervated and regulation of blood flow to the placenta must depend on structural changes and the effect of vasoactive factors. Failure to achieve these adaptations may result in reduced fetoplacental perfusion. The purpose of this study was to determine whether neuronal nitric oxide synthase (nNOS) is expressed in human vascular smooth muscle cells (VSMCs) of the fetoplacental circulation. nNOS has been described as a non-endothelial NOS counterregulating vasoconstriction only in the VSMCs of animal models. Therefore, we investigated nNOS expression in the fetoplacental unit from preeclamptic and healthy pregnancies. METHODS AND RESULTS: We investigated nNOS regulation by immunohistochemistry, Western blotting and reverse transcriptase-polymerase chain reaction analysis. nNOS activity was determined by measuring the conversion of L-3H-arginine to L-3H-citrulline. nNOS expression was revealed only in VSMCs of the human umbilical veins, but not in umbilical arteries. A more direct assessment of nNOS activity showed that a small, but consistent amount of nNOS is present in the denuded media of the umbilical vein. In VSMCs of the umbilical veins during preeclampsia a total loss of nNOS protein expression and a significant decrease in mRNA expression were seen. CONCLUSIONS: Loss of nNOS expression is associated with preeclampsia. It may alter the regulation of blood flow in the fetal and maternal placental vasculature in preeclampsia. However, the impact of NO produced by nNOS on the vascular tone of umbilical veins remains to be elucidated.

In utero exposure to low doses of bisphenol A lead to long-term deleterious effects in the vagina.

The origins of the "endocrine disrupter hypothesis" may be traced to reports on adolescent daughters born to women who had taken the highly potent synthetic estrogen, diethylstilbestrol, while pregnant, and who developed a rare form of vaginal cancer and adenocarcinoma. Bisphenol A (BPA) is an estrogenic chemical that is highly employed in the manufacture of a wide range of consumer products. Some observational studies have suggested that the amounts of BPA to which we are exposed could alter the reproductive organs of developing rodents. We examined the influence of BPA at low doses to address the questions of (a) whether in utero exposure affects the vagina of the offspring and (b) which mechanisms cause the toxic effects. Gravid Sprague-Dawley dams were administered either 0.1 (low dose) or 50 mg/kg per day BPA, the no observed effect level, or 0.2 mg/kg per day 17 alpha-ethinyl estradiol by gavage. Striking morphological changes were observed in the vagina of postpubertal offspring leading us to examine vaginal estrogen receptor (ER) expression because BPA binds to the ER alpha, which is important for growth of the vaginal epithelium. We show that the full-length ER alpha is not expressed during estrus in the vagina of female offspring exposed to either dose of BPA when compared to the control group, whereas ER alpha expression does not differ from the control group during the diestrus stage. ER alpha downregulation seems to be responsible for the observed altered vaginal morphology.

*NO and oxyradical metabolism in new cell lines of rat brain capillary endothelial cells forming the blood-brain barrier.

To investigate the relevance of *NO and oxyradicals in the blood-brain barrier (BBB), differentiated and well-proliferating brain capillary endothelial cells (BCEC) are required. Therefore, rat BCEC (rBCEC) were transfected with immortalizing genes. The resulting lines exhibited endothelial characteristics (factor VIII, angiotensin-converting enzyme, high prostacyclin/thromboxane release rates) and BBB markers (gamma-glutamyl transpeptidase, alkaline phosphatase). The control line rBCEC2 (mock transfected) revealed fibroblastoid morphology, less factor VIII, reduced gamma-glutamyl transpeptidase, weak radical defence, low prostanoid metabolism, and limited proliferation. Lines transfected with immortalizing genes (especially rBCEC4, polyoma virus large T antigen) conserved primary properties: epitheloid morphology, subcultivation with high proliferation rate under pure culture conditions, and powerful defence against reactive oxygen species (Mn-, Cu/Zn-superoxide dismutase, catalase, glutathione peroxidase, glutathione) effectively controlling radical metabolism. Only 100 microM H2O2 overcame this defence and stimulated the formation of eicosanoids similarly as in primary cells. Some BBB markers were expressed to a lower degree; however, cocultivation with astrocytes intensified these markers (e.g., alkaline phosphatase) and paraendothelial tightness, indicating induction of BBB properties. Inducible NO synthase was induced by a cytokine plus lipopolysaccharide mixture in all lines and primary cells, resulting in *NO release. Comparing the cell lines obtained, rBCEC4 are stable immortalized and reveal the best conservation of properties from primary cells, including enzymes producing or decomposing reactive species. These cells can be subcultivated in large amounts and, hence, they are suitable to study the role of radical metabolism in the BBB and in the cerebral microvasculature.

Cerebral endothelial cells release TNF-alpha after stimulation with cell walls of Streptococcus pneumoniae and regulate inducible nitric oxide synthase and ICAM-1 expression via autocrine loops.

TNF-alpha, inducible NO synthase (iNOS), and ICAM-1 are considered to be key proteins in the inflammatory response of most tissues. We tested the hypothesis that cell walls of Streptococcus pneumoniae (PCW), the most common cause of adult bacterial meningitis, induce TNF-alpha, iNOS, and ICAM-1 expression in rat primary brain microvascular endothelial cell cultures. We detected TNF-alpha mRNA by RT-PCR already 1 h after stimulation with PCW, while TNF-alpha protein peaked at 4 h (9.4 +/- 3.6 vs 0.1 +/- 0.1 pg/microgram protein). PCW induced iNOS mRNA 2 h after stimulation, followed by an increase of the NO degradation product nitrite (18.1 +/- 4 vs 5.8 +/- 1.8 at 12 h; 18.1 +/- 4 vs 5.8 +/- 1.8 pmol/microgram protein at 72 h). The addition of TNF-alpha Ab significantly reduced nitrite production to 62.2 +/- 14.4% compared with PCW-stimulated brain microvascular endothelial cells (100%). PCW induced the expression of ICAM-1 (measured by FACS), which was completely blocked by TNF-alpha Ab (142 +/- 18.6 vs 97.5 +/- 12.4%; 100% unstimulated brain microvascular endothelial cells). Cerebral endothelial cells express TNF-alpha mRNA as well as iNOS mRNA and release the bioactive proteins in response to PCW. PCW-induced NO production is mediated in part by an autocrine pathway involving TNF-alpha, whereas ICAM-1 expression is completely mediated by this autocrine loop. By these mechanisms, cerebral endothelial cells may regulate critical steps in inflammatory blood-brain-barrier disruption of bacterial meningitis.

Transgenic rats expressing the human ET-2 gene: a model for the study of endothelin actions in vivo.

Previous studies have characterized the endothelin peptides (ET-1, ET-2, ET-3) as strong vasoconstrictors which are possibly involved in the pathogenesis of cardiovascular disease. Whereas ET-1 and ET-3 have been characterized using a number of approaches, little is known about the function of ET-2. The aim of this study was to define the role of ET-2 in physiology and pathophysiology using a transgenic approach. Transgenic rats expressing a genomic construct of the human ET-2 gene were generated by microinjection of fertilized oocytes from Sprague-Dawley rats. Two transgenic lines were generated, and one line was further characterized in detail. Studies on mRNA expression demonstrated that the transgene is expressed predominantly in kidney, gastrointestinal tract, adrenal gland, lung, and brain. Plasma endothelin levels were elevated 2-fold, and big-endothelin levels were elevated 2.5-fold. Despite these alterations blood pressure in transgenic rats remained normal. Further analysis of transgenic animals revealed that endothelin receptors were not downregulated, and that infusion of exogenous human ET-2 results in an enhanced blood pressure response. These observations suggest the presence of counterregulatory mechanisms influencing the effects of endothelin on blood pressure. One of these mechanisms may involve the nitric oxide system since infusion of an inhibitor of nitric oxide synthase resulted in a greater blood pressure response than in non-transgenic littermates. Despite unchanged blood pressure, alterations were observed in organ development and function, namely of hearts and kidneys, indicating an interference between transgene expression and growth processes. Male rats seem to be more susceptible to endothelin actions. These data show that the elevation in endothelin-2 expression in this transgenic model does not induce hypertension but leads to changes at the end-organ level. Normotension is most likely due to compensatory mechanisms such as increased nitric oxide formation.

Phosphorylation of vasodilator-stimulated phosphoprotein: a consequence of nitric oxide- and cGMP-mediated signal transduction in brain capillary endothelial cells and astrocytes.

There is contradictory information on the relevance of nitric oxide (NO) and cGMP for the function of brain capillary endothelial cells (BCEC) forming the blood-brain barrier (BBB). Therefore, NO/cGMP-mediated signal transduction was investigated in cell cultures of BCEC and of astrocytes (AC) inducing BBB properties in BCEC. Constitutive, Ca2+-activated isoforms of NO synthase (NOS) were found in BCEC (endothelial NOS: eNOS) and in AC (neuronal NOS: nNOS), leading to increased NO release after incubation with the Ca2+-ionophore A23187. Both cell types expressed inducible NOS (iNOS) after incubation with cytokines. Soluble guanylate cyclase (sGC) was detected in both cell types. NO-dependent cGMP formation were observed in BCEC and, less pronounced, in AC. Furthermore, both cell types formed cGMP independently of NO via stimulation of particulate guanylate cyclase (pGC). cGMP-dependent protein kinase (PKG) type Ibeta, but not type II, was expressed in BCEC and AC. In BCEC, vasodilator-stimulated phosphoprotein (VASP) was detected, an established substrate of PKG and associated with microfilaments and cell-cell contacts. Phosphorylation of VASP was intensified by increased intracellular cGMP concentrations. The results indicate that BCEC and, to a smaller degree, AC can form NO and cGMP in response to different stimuli. In BCEC, NO/cGMP-dependent phosphorylation of VASP is demonstrated, thus providing a possibility of influencing cell-cell contacts.

Stretch-activated cation channel in human umbilical vein endothelium in normal pregnancy and in preeclampsia.

OBJECTIVE: To determine whether stretch-activated cation channels (SAC) are present in intact human umbilical vein endothelium (HUVE) and in an endothelial cell line (EA.hy) and whether they act as endothelial mechanosensors, and to determine whether endothelial SAC in HUVE from women with pregnancies complicated by preeclampsia undergo functional changes compared with those in HUVE from women with normotensive pregnancies. METHODS AND RESULTS: By use of the patch-clamp technique we identified a SAC in intact HUVE and in an endothelial cell line. The SAC had mean conductances of 29+/-5 pS (n = 38) for K+ and 12+/-2 pS (n = 4) for Ca2+. Administration of 50 micromol/I gadolinium, a blocker of mechanosensitive ion channels, completely blocked activity of this channel. We found from single-channel recordings that influx of Ca2+ through SAC directly activated high-conductance Ca2+-dependent potassium channels, proving that a significant influx of Ca2+ through SAC occurs at physiologic concentrations of Ca2+. In a comparative study, apparent channel density of SAC (percentage of patches with SAC activity) in HUVE from women with pregnancies complicated by preeclampsia (36.2 +/- 4.3%) was twofold higher than that in HUVE from women with normal pregnancies (17.9+/-2.9%, P< 0.01). Channel conductance and sensitivity to stretching of SAC were not altered by preeclampsia. CONCLUSIONS: Since SAC are capable of acting as endothelial mechanosensors, the greater than normal density of SAC associated with preeclampsia might reflect an alteration of mechanotransduction.

High expression of inducible nitric oxide synthase correlates with intestinal inflammation of interleukin-2-deficient mice.

Severely inflamed colonic sections of IL-2 (-/-) mice showed up to 19-fold increased iNOS mRNA levels. The level of iNOS protein expression corresponded to the increased iNOS mRNA levels as detected by means of Western blot analysis. There was a clear, positive relationship between the level of iNOS expression and the degree of inflammation in the colonic tissue of IL-2 (-/-) and wild-type mice. Our data suggest that iNOS may play a key role in the pathogenesis of ulcerative colitis-like disease in IL-2 (-/-) mice. Further investigation should elucidate the impact of NO on the regulation of the inflammatory process in this model and might contribute to a better understanding of the role iNOS expression in human immune-mediated diseases.

Expression of inducible nitric oxide synthase in placenta of women with gestational diabetes.

Gestational diabetes is one of the most prevalent medical complications of pregnancy and causes increased fetal wastage. Investigation of placentas from diabetic mothers indicate chronic disturbances in intervillous, circulation, dilatation of capillaries, and a relatively immature villous structure. Abnormal levels of nitric oxide (NO) may contribute to maternal disorders such as the pathogenesis of diabetic vascular complications. In the normal placenta NO is generated only by endothelial NOS, which apparently serves to regulate vascular tone in the fetoplacental circulation. In contrast, studies have reported the absence of inducible nitric oxide synthase (iNOS) in human placentas under normal conditions. The aim of our study was to investigate whether iNOS is expressed in placentas from patients with gestational diabetes. Reverse transcription-polymerase chain reaction and Western blot analysis demonstrated iNOS mRNA and protein expression in placental tissue only from patients with gestational diabetes. Immunohistochemistry localized iNOS staining to endothelial cells and trophoblasts. We conclude that iNOS can be expressed in human placenta. Its expression might play an important role in placental pathophysiology.

Role of the cardiac renin-angiotensin system in human heart failure.

The local effects of angiotensin II (ANG II) on the heart may play an important role for the pathophysiology of cardiovascular disease. Numerous in vitro studies have demonstrated that angiotensin II has distinctive cellular effects in the cardiovascular system which are independent from its effects on blood pressure. These have led to the hypothesis that activation of the angiotensin system in the heart could be of functional relevance for the adaptive processes in several cardiovascular disorders such as cardiac hypertrophy heart failure. This concept has been further supported by clinical studies showing the beneficial effects of angiotensin-converting enzyme inhibitors in these circumstances. In order to study the gene regulation of renin-angiotensin system components in cardiac disorders we investigated the gene expression of angiotensin converting enzyme in human heart failure. Results showed that the enzyme is activated locally in this condition, supporting previous studies in animals. Taken together with recent evidence from genetic studies linking the enzyme to myocardial infarction and cardiac hypertrophy, our findings are in support of the notion that angiotensin converting enzyme plays a central role in cardiovascular physiology and pathophysiology.

The perivascular contractile sheath of human placental stem villi: its isolation and characterization.

Former studies have shown that fetal blood vessels of stem villi in the human placenta are enclosed by sheaths of presumed contractile cells. Efforts to prove the contractility of these sheaths by isometric and biochemical investigations still suffer from the difficulty of differentiating between extravascular and vascular (media) contractile cells. The present study describes a method for the selective dissection of perivascular tissue sheaths in stem villi of approximately 2-4 mm thickness, which contained abundant alpha-actin immunoreactive cells and were free of adherent vascular smooth muscle cells. These extravascular contractile cells are part of the 'fibrous paravascular sheath', which, in placental pathology, is used as an index of maturity. To emphasize the high number of contractile cells and their location within this sheath, we propose the common term perivascular contractile sheath (PVCS). The isolation method offers the possibility of selectively investigating the contractile forces of the PVCS and of obtaining more insight into its functional role.

Regulation of the endothelin system in transgenic rats expressing the human endothelin-2 gene.

We have established a transgenic rat model for the expression of the human endothelin-2 (ET-2). These animals exhibit overexpression of the transgene in tissues as well as in plasma. Despite these changes, blood pressure remains normal. To understand the regulatory mechanisms for normotension in the presence of increased ET-2 levels, we have investigated the ET system in more detail. We used competitive reverse transcription-polymerase chain reaction (RT-PCR) to evaluate possible overexpression or downregulation of endothelin A and B receptors at the mRNA level. PCR analyses revealed no significant differences of ETA and ETB receptor expression. In conclusion, the expression of human ET-2 in transgenic rats does not result in hypertension. Normotension in the transgenic animals is independent of ET receptor regulation. The reason for this may be counterregulation by other vasoactive systems, such as the NO system. Future studies will take this into account and will also concentrate on possible histomorphologic alterations caused by mitogenic properties of the endothelins.

The extravascular contractile system in the human placenta. Morphological and immunocytochemical investigations.

In the human placenta, besides the fetal blood vessel system a second extravascular contractile system exists. It is localized in the chorionic plate and runs in a longitudinal direction and adjacent to fetal blood vessels into the stem villi, where it forms perivascular contractile sheaths. Characteristically, cells of the extravascular contractile system are extremely long and spindle-shaped and give rise to fine cell processes, by which they obviously contact each other or insert into the basement membrane of the trophoblast. They show immunoreactivity with desmin, vimentin, alpha-actin, myosin, nitric oxide synthase type I (brain form) and dipeptidyl peptidase IV. The ultrastructure suggests that cells of the extravascular contractile system are related to smooth muscle cells, including subpopulations with myofibroblastic features. In stem villi a few cells are nitric oxide synthase type I immunoreactive. These cells are thought to be specialized smooth-muscle-like cells of the extravascular contractile system or cells of the extravascular contractile system related to paraneurons that generate nitric oxide, which, in turn, may modulate the tone of perivascular contractile sheaths. The high dipeptidyl peptidase IV activity suggests that modulation of the extravascular contractile system may also occur by substance P.

Distribution of constitutive nitric oxide synthase immunoreactivity and NADPH-diaphorase activity in murine telogen and anagen skin.

The freely diffusible radical nitric oxide is generated by nitric oxide synthase, and is a pleiotropic, bioregulatory molecule that regulates, e.g., the vascular tone, functions as a major neurotransmitter, and is involved in macrophage-mediated cytotoxicity and platelet aggregation. Constitutive nitric oxide synthase exhibits NADPH-diaphorase activity that can be demonstrated histochemically. To study whether this enzyme is present in mammalian skin during distinct phases of the murine hair cycle, we have examined cryosections of C 57 BL-6 mouse skin in telogen and depilation-induced anagen VI. Histochemical analysis of NADPH-diaphorase activity was complemented by immunohistology, using two specific rabbit antisera against constitutive neuronal nitric oxide synthase. Epidermis and the outer root sheath showed both immunoreactivity for the enzyme and NADPH-diaphorase activity, whereas dermal papilla and sebaceous glands displayed only strong NADPH-diaphorase activity, suggesting that this enzyme histochemical test measures additional enzymes besides nitric oxide synthase. Intrinsic nitric oxide synthase immunoreactivity was also detected by immunoblot in mouse skin homogenates, staining proteins of an apparent 160-kDa molecular weight. Compared to telogen skin, these immunoreactive proteins were quantitatively increased in anagen VI skin. Thus, our study suggests that defined epithelial compartments of normal murine skin are capable of synthesizing nitric oxide and that the molecule may be involved in skin physiology, growth, and remodeling.

[The extravascular contractile system of the human placenta: a new aspect for function of the organ?]. / Das extravasale kontraktile System der Humanplazenta: Ein neuer Aspekt für die Funktion des Organs?

In the chorionic plate and stem villi of the human placenta besides the fetal blood vessel system a second extravascular contractile system (EVCS) exists. The cells of this system contain contractile and intermediate filaments and are dipeptidyl peptidase-IV- and NO-synthase-type-I-(NOS)-immunoreactive. Therefore it can be assumed that these cells cleave the vasodilator Substance P (by DPP IV) and produce NO (by NOS) and may contribute to a modulation of the EVCS.

Etiology and incidence of acute pancreatitis: a 20-year study in a single institution.

Reports from various countries raise the question of changes in the incidence and etiology of acute pancreatitis. This study covers patients admitted with acute pancreatitis to a single university department of medicine between 1965 and 1970 and between 1980 and 1985. The absolute number of patients admitted with acute pancreatitis doubled, whereas the percentage of pancreatitic patients in the total population of admitted patients decreased by 38%. No firm conclusion can be made about an increasing incidence of acute pancreatitis from 1970 to 1985. However, the dominating etiological factor was no longer biliary tract disease but significantly shifted to alcoholism, whereas changes in the severity of the disease were not significant. There was a downward trend in mortality with mortality rates of 13 and 8.6% for the first and second period, respectively; however, because of the small number of patients this change did not reach the level of significance.

[Mineralocorticoid-induced changes of vascular reactivity to noradrenaline and angiotensin II in baboons]. / Mineralokortikoidbedingte Veränderung der vaskulären Reaktivität auf Noradrenalin und Angiotensin II bei Pavianen.

In experiments on normotensive baboons of both sex the vascular reactivity on angiotensin II and noradrenaline was studied after an 18-week administration of DOCA (2 mg/b.w./week i. m.) and of common sal (5 g/day with meals). Dosage and duration of DOCA administration was chosen to prevent hypertensive haemodynamic alterations. 40 to 240 seconds after an angiotensin II injection (0.5 micrograms/kg b.w.) the pressor, especially diastolic response in both narcotized and non-narcotized animals intensified markedly. Infusion of noradrenaline (1.0 micrograms/kg b.w./min for 5 min) called forth after 120 min a significant rise in diastolic BP. Described abnormal pressor response is considered to represent a suitable model of a vascular predisposition to hypertension.