Insights into possibilities for grouping and read-across for nanomaterials in EU chemicals legislation.

This paper presents a comprehensive review of European Union (EU) legislation addressing the safety of chemical substances, and possibilities within each piece of legislation for applying grouping and read-across approaches for the assessment of nanomaterials (NMs). Hence, this review considers both the overarching regulation of chemical substances under REACH (Regulation (EC) No 1907/2006 on registration, evaluation, authorization, and restriction of chemicals) and CLP (Regulation (EC) No 1272/2008 on classification, labeling and packaging of substances and mixtures) and the sector-specific pieces of legislation for cosmetic, plant protection and biocidal products, and legislation addressing food, novel food, and food contact materials. The relevant supporting documents (e.g. guidance documents) regarding each piece of legislation were identified and reviewed, considering the relevant technical and scientific literature. Prospective regulatory needs for implementing grouping in the assessment of NMs were identified, and the question whether each particular piece of legislation permits the use of grouping and read-across to address information gaps was answered.

A case of idiopathic tracheal stenosis.

Acquired tracheal stenosis is a condition that often results from trauma, neoplasm, infection, vasculitis, inflammatory or infiltrative processes. Idiopathic tracheal stenosis is a rare entity and represents a diagnosis of exclusion. We report a case of severe localized extrathoracic tracheal stenosis in a 35 year old female who was initially suspected to have asthma as the cause of several months of chronic dyspnea.

Melatonin protection against burn-induced hepatic injury by down-regulation of nuclear factor kappa B activation.

Melatonin exhibits a wide variety of biological activity including antioxidant and anti-inflammatory effects. We have previously reported its protective effect on hepatic oxidative hepatic injury in burns. In this study, we investigated the role of nuclear factor kappa B (NF-κB) in melatonin-mediated protection against liver injury by using the burned-rat model. Melatonin (N-acetyl-5-methoxytriptamin, 10mg/kg (-1), i.p.) was administered immediately and 12 hours after thermal skin injury. Hepatic NF-κB expression was determined by Western blotting. TNF-α level in liver homogenate was quantified using enzyme-linked immunosorbent assay (ELISA) kit. Plasma aspartate aminotransferase (AST) and alanine aminotransferase (ALT) levels were determined to assess liver injury at the 24th hour after burns. Thermal skin injury caused significant elevation of hepatic NF-κB expression by 48 percent, TNF-α level by 55 percent and plasma AST and ALT activities by 2- and 3-fold, respectively, in comparison with normal control rats. Treatment with melatonin decreased significantly elevated hepatic NF-κB activity and TNF-α, maintaining the levels close to the control values Melatonin suppressed the elevation of plasma AST and ALT activities (p less than 0.001), which remained significantly increased compared to controls. In conclusion, thermal skin injury causes hepatic NF-κB activation that may mediate the release of hepatic TNF-α and contribute to liver damage. Melatonin protects against burn-induced hepatic injury as to a certain extent this effect may result from the suppression of NF-κB-mediated inflammatory response.

[Asymmetric obstructed uterus didelphys/incomplete Herlyn-Werner-Wunderlich syndrome/diagnosed by transvaginal 3D ultrasound--a case report].

Asymmetric obstructed uterus didelphys (Herlyn-Werner-Wunderlich syndrome-HWWS) is a rare congenital Müllerian anomaly consisting of uterus didelphys, hemivaginal septum and ipsilateral renal agenesis. We present a case of an incomplete HWWS diagnosed by 3D transvaginal ultrasound in a 22 year old patient with absence of the hemivaginal septum. The most contributive diagnostic factors as well as the appropriate therapeutic management in such cases are discussed.

Cholinergic responses of ileal longitudinal muscle under short-lasting exposure to cupric ions.

1 The effect of short-term exposure to cupric ions (Cu2+) on electric field-stimulated (EFS) or agonist-induced contractions of guinea-pig isolated ileum was studied. 2 EFS elicited tetrodotoxin- and atropine-sensitive contractions that were concentration dependently inhibited by Cu2+ (IC50 = 14.7 +/- 4.2 microm). Maximal inhibition (90.4 +/- 3.1% of baseline contractions) was attained with 30 microm Cu2+. 3 Carbachol induced concentration-dependent contractions (EC50 = 0.021 +/- 0.004 microm) that were inhibited by 0.3 microm atropine to a non-competitive manner (decreased maximal response, EC50 value = 0.26 +/- 0.04 microm, K(e) = 0.026 microm). Cu2+ (15 microm) potentiated contractions induced by carbachol, such that the maximum response was increased by 30.3 +/- 10.4%. 4 Histamine induced concentration-dependent contractions of the longitudinal muscle (EC50 = 0.11 +/- 0.03 microm). Dyphenhydramine (0.1 microm) decreased the maximum response to histamine and shifted the curve to the right (EC50 value = 4.71 +/- 0.35 microm, K(e) = 0.0024 microm). Cu2+ (15 microm) caused a rightward shift of the histamine concentration-response curve (EC50 = 0.61 +/- 0.1 microm) without changing the maximum response. Serotonin induced concentration-dependent contractions at concentrations higher than 10 nM (EC50 value of 0.34 +/- 0.12 microm) were not significantly affected by 15 microm Cu2+. 5 Our results suggest that in ileal longitudinal muscle, Cu2+ inhibits cholinergic neurotransmission but also facilitates postsynaptic muscarinic receptor responses.

High-glucose-induced metallothionein expression in endothelial cells: an endothelin-mediated mechanism.

Vascular endothelial cells are constantly exposed to oxidative stress and must be protected by physiological responses. In diabetes mellitus, endothelial cell permeability is impaired and may be increased by high extracellular glucose concentrations. It has been postulated that metallothionein (MT) can protect endothelial cells from oxidative stress with its increased expression by cytokines, thrombin, and endothelin (ET)-1. In this study, we demonstrate that high glucose concentration can induce MT expression in endothelial cells through a distinct ET-dependent pathway. Exposure of human umbilical vein endothelial cells (HUVEC) to increasing concentrations of glucose resulted in a rapid dose-dependent increase in MT-2 and ET-1 mRNA expression. MT expression may be further augmented with addition of ET-1. Preincubation of the cells with the specific ET(B) antagonist BQ-788 blocked MT-2 mRNA expression more effectively than the ET(A) inhibitor TBC-11251. High glucose also increased immunoreactive MT protein expression and induced translocation of MT into the perinuclear area. Perinuclear localization of MT was related to high-glucose-induced reorganization of F-actin filaments. These results demonstrate that an increase in extracellular glucose in HUVEC can lead to a rapid dose-dependent increase in MT-2 mRNA expression and to perinuclear localization of MT protein with changes to the cytoskeleton. These effects are mediated via the ET receptor-dependent pathway.

Interaction of endothelin-1 with vasoactive factors in mediating glucose-induced increased permeability in endothelial cells.

Alteration of endothelins (ET) and/or their receptors may be important in mediating vascular dysfunction in diabetes. We investigated mechanisms regulating ET-1 expression in human umbilical vein endothelial cells (HUVEC) in response to glucose and the functional significance of these mechanisms. Permeability across HUVEC, grown in medium containing either low (5 mmol/l) or high (25 mmol/l) D-glucose were investigated. L-glucose was used as a control. ET-1, ET(A), and ET(B) mRNA were assessed by semiquantitative RT-PCR. ET-1 immunoreactivity and F-actin microfilament assembly were investigated using confocal microscopy. Increased transendothelial permeability was noted in cells cultured in high glucose or when the cells grown in low (physiologic) glucose were incubated with ET-1, vascular endothelial growth factor (VEGF), or N (G) -nitro-L-arginine methyl ester but not when they were incubated with ET-3, N(G)-nitro-D-arginine methyl ester, or L-glucose. Increased permeability was associated with increased ET-1, ET(A), and ET(B) mRNA expression and augmented ET-1 immunoreactivity. High glucose induced increased permeability, increased ET-1, ET(A), and ET(B) mRNA expression. ET-1 immunoreactivity was blocked by the protein kinase C (PKC) inhibitor chelerythrine, the specific PKC isoform inhibitor 379196, VEGF-neutralizing antibody, or the ET(A) blocker TBC11251, but was not blocked by the specific ET(B) blocker BQ788 or by a VEGF-non-neutralizing antibody. Increased permeability was also associated with deranged F-actin assembly in the endothelial cells and by derangement of endothelial cell junctions as assessed by electron microscopy. Data from this study suggest that high glucose-induced increased permeability may be induced through increased ET-1 expression and disorganization of F-actin assembly. ET-1 expression and increased permeability may occur secondary to PKC isoform activation and may be modulated by VEGF and nitric oxide.

Nuclear localization of metallothionein during cell proliferation and differentiation.

Although MT is detected as a cytoplasmic protein in hepatocytes of adult liver, it can be localized in the hepatocyte nuclei in human fetal liver bound to zinc and copper. Both nuclear and cytoplasmic localization of MT have been observed in several human tumours, especially in regions of high proliferation. Transient co-localization of zinc and MT has been shown in differentiating myoblast and 3T3-L1 fibroblasts, and during the G1-/S-phase progression in cell cycle. Several mechanisms have been proposed for the import and retention of MT in the nucleus, including signal transduction pathways. The high levels of MT in the nucleus of cells under certain conditions may be related to the increased requirement for zinc for several metallo-enzymes and transcription factors during rapid growth. The function of nuclear MT may be to protect the cell from DNA damage and apoptosis, and also to regulate gene expression during certain stages of cell cycle.

Astrocyte cultures from transgenic mice to study the role of metallothionein in cytotoxicity of tert-butyl hydroperoxide.

The cell viability, lipid peroxidation (LPO) and hydrogen peroxide (H(2)O(2)) generation were measured in cultured primary astrocytes, from metallothionein (MT)-I isoform overexpressing transgenic (MT-I*), MT-I/MT-II null and control mice after exposure to tert-butylhydroperoxide (tBH). Astrocytes from MT-I* mice have high basal levels of both MT-I mRNA and MT protein, whereas there is only MT-III isoform in astrocytes from MT-I/MT-II null mice. The results showed that (1) cultured astrocytes from MT-I* mice were most resistant to the cytotoxicity of tBH and those from MT-I/MT-II null mice were most sensitive to the cytotoxicity of tBH; (2) LPO after exposure to tBH were increased in all cells, but the levels were the highest in astrocytes from MT-I/MT-II null mice, while those in MT-I* mice were the lowest; (3) the levels of H(2)O(2) in cultured astrocytes from MT-I* mice were the lowest, while those in astrocytes from MT-I/MT-II null mice were the highest. These results support the hypothesis that MT can scavenge free radicals and protect astrocytes from oxidative stress.

Signal transduction pathways, and nuclear translocation of zinc and metallothionein during differentiation of myoblasts.

The changes in subcellular localization of metallothionein during differentiation were studied in two myoblast cell lines, L6 and H9C2. Addition of insulin like growth factor-I or lowering foetal bovine serum to 1% can induce differentiation of myoblasts to myotubes. Metallothionein and zinc were localized mainly in the cytoplasm in myoblasts but were translocated into the nucleus of newly formed myotubes during early differentiation. In fully differentiated myotubes, metallothionein content was decreased with a cytoplasmic localization. Addition of an inhibitor of mitogen-activated protein kinase, PD 98059, did not affect differentiation but blocked nuclear translocation of metallothionein. LY 294092, an inhibitor of PI3 kinase, and rapamycin, an inhibitor of p70S6 serine/threonine kinase, abolished insulin-like growth factor-I induced differentiation of myoblasts, retained metallothionein in the cytoplasm, and decreased metallothionein content. These results demonstrate that the cytoplasmic-nuclear translocation of metallothionein occurs during the early stage of differentiation of myoblasts to myotubes and can be blocked by inhibition of certain signal transduction pathways. The transient nuclear localization of metallothionein and zinc may be related to a high requirement for zinc for metabolic activities during the early stage of differentiation.

Delay of M-phase onset by aphidicolin can retain the nuclear localization of zinc and metallothionein in 3T3-L1 fibroblasts.

The transient nuclear localization of metallothionein during cell growth and differentiation may be related to the increased requirement of zinc for DNA synthesis, activation of metalloenzymes, and transcription factors. Treatment of 3T3-L1 fibroblasts with aphidicolin, an inhibitor of nuclear DNA synthesis, caused a cell-cycle block at G1/S phase and a delay in the onset of M phase. This also resulted in the accumulation of both zinc and metallothionein in the nucleus. After removal of aphidicolin, the cells rapidly reentered S phase, and during the G2/M phase of cell cycle both zinc and metallothionein began to relocate to the cytoplasm. Delaying the onset of M phase in 3T3-L1 cells could prevent the cytoplasmic relocation of metallothionein. The nuclear translocation of both zinc and metallothionein during the cell cycle can be considered as a normal process and this may be a general mechanism in response to mitogenic signals.

Metallothionein and apoptosis during differentiation of myoblasts to myotubes: protection against free radical toxicity.

The changes in subcellular localization of metallothionein (MT) during differentiation were studied in two muscle cell lines, L6 and H9C2, myoblasts in order to understand the nuclear presence of MT and its antiapoptotic property. In myoblasts, MT and zinc were localized mainly in the cytoplasm but were translocated into the nucleus of newly formed myotubes during early stage of differentiation, which was initiated by lowering FBS from 10% to 1%. In fully differentiated myotubes, metallothionein content was decreased with a cytoplasmic localization. These changes in subcellular localization of MT and Zn were accompanied by increased apoptosis in myotubes. The changes in the apoptosis at different stages of differentiation were measured by both DNA ladder formation and TUNEL technique. The results also show that the apoptosis may be initiated by free radical generation and may be accompanied by p53 expression. The H9C2 cells contained high levels of MT, differentiated slowly, and had low incidence of apoptotic bodies compared to L6 cell line.

New competitive enzyme-linked immunosorbent assay for determination of metallothionein in tissue and sera.

Very little information is available concerning the relationship between metallothionein (MT) and diseases in humans. Several methods to measure MT levels exist but many of these assays are not sensitive to measure MT in human sera. A new sensitive competitive ELISA system has been developed using MT labeled with horseradish peroxidase as a conjugate and high-titre polyclonal antibodies obtained from rabbit immunoglobulin G for MT determination in human sera. The cELISA proposed here permits a reliable determination of MT in the range 10-2 000 000 pg ml(-1). The method was compared with Cd-hem assay and showed good agreement of results. The recovery of the assay was determined by spiking rat MT into rat and human sera, and comparing it with spiked diluent controls. The overall recoveries of the added MT were 101% for rat sera and 89% for human sera. The variation within-assay and between assay were 3 and 6%, respectively. A significant difference (P<0.001) was found between the MT-level in human sera from patient with essential hypertension (646+/-223 ng ml(-1), n=90) and normotensive subjects (21+/-18 ng ml(-1), n=236). A correlation between arterial hypertension and MT-level seems possible. A very sensitive new cELISA method was presented for determination of MT in sera and tissues. It enables investigation of possible correlations between sera MT-concentration and certain diseases.

Analysis of the possible protective role of metallothionein in streptozotocin-induced diabetes using metallothionein-null mice.

In order to clarify a possible protective role of metallothionein (MT) in the development of streptozotocin (STZ)-caused insulin-dependent diabetes mellitus (IDDM) and its mechanisms, we studied whether MT is effective for protection against STZ-caused IDDM by utilizing MT-null (isoforms MT-I and II) transgenic mice. It was found that Zn pretreatment (I mg/kg body weight as ZnSO4) has a unique inhibitory effect on IDDM development in MT-null mice in contrast to no marked effect in control (C57BL/6J) mice, suggesting that Zn ions free from MT molecules exerted this protective effect. The highest Zn dose (10 mg/kg body weight) fully suppressed development of hyperglycaemia in both types of mice. Pretreatment with Zn partially led to recovery of superoxide dismutase activities in the liver and pancreas in which STZ administration suppressed superoxide dismutase activity in both types of mice. The present study suggests that Zn plays an important role in the pathogenesis of IDDM, although a possible involvement of MT in the protection of STZ-caused IDDM cannot be completely negated.

Susceptibility of metallothionein-null mice to paraquat.

Using transgenic mice in which metallothionein (MT)-I and MT-II genes, we have studied a putative role of MT as a free radical scavenger against paraquat, a free radical generator. Male mice were injected s.c. with paraquat (PQ) at a single dose of 40 or 60 mg/kg of body weight (b.w.). Two of the six MT-null mice died within 16 h at the dose of 60 mg PQ/kg. b. w. PQ administration increased hepatic MT concentration in the normal mice (C57BL/6J), but not in the MT-null mice. The lipid peroxidation (LP) determined by thiobarbituric acid-reactive substance formation was increased by PQ in the liver of normal and MT-null mice, and the enhanced level was greater in the MT-null mice than in the C57BL/6J mice. Administration of PQ significantly increased blood urea nitrogen only in the MT-null mice, indicating renal damage. Without paraquat administration, the hepatic concentration of non-protein sulphydryl compounds was less in the MT-null mice than in the C57BL/6J mice, and the basal level of LP was higher in the MT-null mice than in the C57BL/6J mice. The present results support the notion that MT plays an antioxidative role against paraquat insult under physiological conditions.

Apometallothionein in rat liver.

The identification of apometallothionein (AMT) in rat liver by reversed-phase high-performance liquid chromatography (RP-HPLC) after gel permeation was realized in experiments performed both in vivo and in vitro. The reliable assignment of the corresponding AMT peak permitted the detection and determination of AMT in different groups of experimental and control rats. In all animals studied (more than 100 rats), AMT was always present in amounts higher than that of metallothionein (MT) or compatible with it. Induction of MT synthesis by CdCl2 subcutaneous injections decreased the AMT level and increased the MT level, but nevertheless the amount of AMT still remained relatively high.

Metallothioneins in spontaneously hypertensive rat liver.

The metallothionein (MT) synthesis was induced in the liver of spontaneously hypertensive rats (SHR) and Wistar Kyoto (WKY) rats through sc injections of CdCl2 for 3 and 6 days. The MT contents of the liver of these animals and of untreated rats from both groups were determined by gel filtration, HPLC, SDS/PAGE and amino acid analysis. The isoforms MT1 and MT2 were identified and their Cd, Zn and SH-group contents were determined. The SHR showed significantly higher values of MT than WKY rats in the untreated animals and on the 3rd day of the induction. On the 6th day, the MT levels in both groups were equal. The Cd and Zn contents followed the MT concentration in the homogenates. The possible relation between the arterial hypertension and the zinc and copper homeostasis is discussed.

Attraction, deformation and contact of membranes induced by low frequency electric fields.

The force of attraction between erythrocyte ghosts induced by low frequency electric fields (60 Hz) was measured as a function of the intermembrane separation. It varied from 10(-14) N for separation of the order of the cell diameter to 10(-12) N for close approach and contact in 20 mM sodium phosphate buffers (conductivity 260 mS/m, pH 8.5). For large separations the interaction force followed a dependence on separation as predicted for dipole-dipole interactions. For small separation an empirical formula was obtained. The membranes deformed at close approach (less than 1 microns) before making contact. The contact area increased with time until reaching the final equilibrium state. The ghosts separated reversibly after switching off the electric field. The membrane tension induced by the ghost interaction at contact was estimated to be of the order of 0.1 mN/m. These first quantitative measurements of the force/separation dependence for intermembrane interactions induced by low frequency electric fields indicate that attractive forces, membrane deformation and contact area of cells depend strongly on intermembrane separation and field strength. The quantitative relationship between them are important for measuring membrane surface and mechanical properties, intermembrane forces and understanding mechanisms of membrane adhesion, instability and fusion in electric fields and in general.