Burden of disease and costs of exposure to endocrine disrupting chemicals in the European Union: an updated analysis.

A previous report documented that endocrine disrupting chemicals contribute substantially to certain forms of disease and disability. In the present analysis, our main objective was to update a range of health and economic costs that can be reasonably attributed to endocrine disrupting chemical exposures in the European Union, leveraging new burden and disease cost estimates of female reproductive conditions from accompanying report. Expert panels evaluated the epidemiologic evidence, using adapted criteria from the WHO Grading of Recommendations Assessment, Development and Evaluation Working Group, and evaluated laboratory and animal evidence of endocrine disruption using definitions recently promulgated by the Danish Environmental Protection Agency. The Delphi method was used to make decisions on the strength of the data. Expert panels consensus was achieved for probable (>20%) endocrine disrupting chemical causation for IQ loss and associated intellectual disability; autism; attention deficit hyperactivity disorder; endometriosis; fibroids; childhood obesity; adult obesity; adult diabetes; cryptorchidism; male infertility, and mortality associated with reduced testosterone. Accounting for probability of causation, and using the midpoint of each range for probability of causation, Monte Carlo simulations produced a median annual cost of €163 billion (1.28% of EU Gross Domestic Product) across 1000 simulations. We conclude that endocrine disrupting chemical exposures in the EU are likely to contribute substantially to disease and dysfunction across the life course with costs in the hundreds of billions of Euros per year. These estimates represent only those endocrine disrupting chemicals with the highest probability of causation; a broader analysis would have produced greater estimates of burden of disease and costs.

Diagnostics and therapy of muscle channelopathies--Guidelines of the Ulm Muscle Centre.

This article is dedicated to our teacher, Prof. Erich Kuhn, Heidelberg, on the occasion of his 88th birthday on 23rd November 2008. In contrast to muscular dystrophies, the muscle channelopathies, a group of diseases characterised by impaired muscle excitation or excitation-contraction coupling, can fairly well be treated with a whole series of pharmacological drugs. However, for a proper treatment proper diagnostics are essential. This article lists state-of-the-art diagnostics and therapies for the two types of myotonic dystrophies, for recessive and dominant myotonia congenita, for the sodium channel myotonias, for the primary dyskalemic periodic paralyses, for central core disease and for malignant hyperthermia susceptibility in detail. In addition, for each disorder a short summary of aetiology, symptomatology, and pathogenesis is provided.

Topology of the human skeletal muscle chloride channel hClC-1 probed with hydrophilic epitope insertion.

To investigate the membrane topology of the skeletal muscle chloride channel ClC-1, we inserted the small antigenic flag (DYKDDDDK) and/or HSV (QPELAPEDPED) epitope tags into nine predicted extra- and intracellular loops along the channel protein. Functional integrity of the modified proteins was tested by measuring the chloride currents conducted by these channels expressed in tsA201 cells. Insertion of the tags into the linkers D1D2, D4D5, D6D7, D8D9 or D11D12 did not alter channel function significantly, whereas insertion into D3D4, D5D6, D9D10 and D10D11 led to loss of function. Intra- or extracellular localisation of the tags was determined by immunofluorescent staining of intact and permeabilised tsA201 cells transiently transfected with the functional epitope-inserted constructs. Intact cells stained for the epitope tags inserted into D1D2, D6D7 and D8D9, indicating that these linkers face the extracellular side of the membrane. No conclusions could be drawn for the location of D4D5 and D11D12. Insertion of the flag epitope at position P260 (linker D4D5), a putative pore-lining region, did not change any of the channel function properties markedly, suggesting that the region surrounding P260 cannot directly line the ion conduction pathway of ClC-1.

The precautionary principle in environmental science.

Environmental scientists play a key role in society's responses to environmental problems, and many of the studies they perform are intended ultimately to affect policy. The precautionary principle, proposed as a new guideline in environmental decision making, has four central components: taking preventive action in the face of uncertainty; shifting the burden of proof to the proponents of an activity; exploring a wide range of alternatives to possibly harmful actions; and increasing public participation in decision making. In this paper we examine the implications of the precautionary principle for environmental scientists, whose work often involves studying highly complex, poorly understood systems, while at the same time facing conflicting pressures from those who seek to balance economic growth and environmental protection. In this complicated and contested terrain, it is useful to examine the methodologies of science and to consider ways that, without compromising integrity and objectivity, research can be more or less helpful to those who would act with precaution. We argue that a shift to more precautionary policies creates opportunities and challenges for scientists to think differently about the ways they conduct studies and communicate results. There is a complicated feedback relation between the discoveries of science and the setting of policy. While maintaining their objectivity and focus on understanding the world, environmental scientists should be aware of the policy uses of their work and of their social responsibility to do science that protects human health and the environment. The precautionary principle highlights this tight, challenging linkage between science and policy.

CSF filtration is an effective treatment of Guillain-Barré syndrome: a randomized clinical trial.

OBJECTIVE: To compare CSF filtration (CSFF) and plasma exchange (PE) in the treatment of patients with Guillain-Barré syndrome (GBS). METHODS: In a prospective controlled clinical trial, 37 patients with acute GBS were randomized to receive either CSFF or PE. Inclusion criteria were fulfillment of National Institute of Neurological and Communicative Disorders and Stroke criteria and disability to walk >5 m unassisted. RESULTS: With similar baseline features in both groups (initial disability grades on the six-point grading scale of the GBS Study Group) the primary outcome variable (improvement within 28 days after randomization) was almost identical (test for equivalence p = 0.0014), the mean grade values being 0.82 in the CSFF group and 0.80 in the PE group. After 56 days, 56% (9 of 16 patients) of the CSFF group and 37% (7 of 19 patients) of the PE group had reached grade 2 (i.e., ability of unassisted walking >5 m). After 6 months, the probability to reach grade 2 was about 80% in both groups. In the CSFF group, transient pleocytosis occurred without apparent clinical complications. Clinically relevant complications were higher in the PE-treated group. CONCLUSIONS: Although the number of patients was small, the authors found that the treatment of GBS with CSFF is at least as effective as with PE. CSFF might work by removing from the CSF inflammatory mediators, autoantibodies, or other factors.

Staircase-like potentiation of calcium release in mouse myotubes during repetitive short-term application of threshold caffeine.

The release of Ca2+ in response to caffeine at threshold concentration (5 mM) was studied in mouse skeletal myotubes. Repeated 5-s applications of caffeine, each followed by a 30-s washout, caused Ca2+ releases of consecutively growing amplitude (staircase phenomenon). Each response declined rapidly and had a slow tail. Repeated applications of threshold caffeine lowered the threshold concentration. The interval between threshold applications could be increased to 30 min without loss of potentiation. When threshold caffeine was applied continuously for up to 10 min, the increase in Ca2+ concentration as seen with staircase potentiation did not occur. Depolarization by elevated [K+] or by voltage-clamp steps potentiated caffeine-induced Ca2+ release rapidly as compared to the slow exponential growth of staircase-like potentiation. Gd3+ prevented the depolarization-induced potentiation, but not the staircase phenomenon. Staircase-like potentiation of Ca2+ release was evident even when the voltage sensors were clamped in their resting state; in contrast, potentiated Ca2+ release and its rapid termination apparently require conversion of the voltage sensors to an activated state. Staircase potentiation was blocked when Ca2+ was omitted from the bath, thus pinpointing the source of Ca2+. We suggest that staircase-like potentiation is conditioned by a caffeine-dependent Ca2+ influx across the plasma membrane.

Identification of selected hormonally active agents and animal mammary carcinogens in commercial and residential air and dust samples.

In order to characterize typical indoor exposures to chemicals of interest for research on breast cancer and other hormonally mediated health outcomes, methods were developed to analyze air and dust for target compounds that have been identified as animal mammary carcinogens or hormonally active agents and that are used in commercial or consumer products or building materials. These methods were applied to a small number of residential and commercial environments to begin to characterize the extent of exposure to these classes of compounds. Phenolic compounds, including nonylphenol, octylphenol, bisphenol A, and the methoxychlor metabolite 2,2-bis(p-hydroxyphenyl)-1,1,1-trichloroethane (HPTE), were extracted, derivatized, and analyzed by gas chromatography/mass spectrometry (GC/MS)-selective ion monitoring (SIM). Selected phthalates, pesticides, polycyclic aromatic hydrocarbons (PAHs), and polychlorinated biphenyls (PCBs) were extracted and analyzed by GC/MS-SIM. Residential and workplace samples showed detectable levels of twelve pesticides in dust and seven in air samples. Phthalates were abundant in dust (0.3-524 micrograms/g) and air (0.005-2.8 micrograms/m3). Nonylphenol and its mono- and di-ethoxylates were prevalent in dust (0.82-14 micrograms/g) along with estrogenic phenols such as bisphenol A and o-phenyl phenol. In this 7-sample pilot study, 33 of 86 target compounds were detected in dust, and 24 of 57 target compounds were detected in air. In a single sample from one home, 27 of the target compounds were detected in dust and 15 in air, providing an indication of chemical mixtures to which humans are typically exposed.

Optical detection of mitochondrial NADH content in intact human myotubes.

Time-gated fluorescence spectroscopy in combination with non-radiative energy transfer was used on intact human skeletal myotubes for the determination of the mitochondrial NADH content which is considered to be a sensitive indicator of mitochondrial function. To mimic dysfunction of the mitochondrial energy metabolism, complexes I or III of the respiratory chain were inhibited by drugs. In the absence of the fluorescent mitochondrial marker rhodamine (R123), the NADH autofluorescence (i.e. a signal monitoring cytoplasmic plus mitochondrial NADH) remained unchanged upon inhibition of complex I by rotenone, and was increased by a factor of 2 upon inhibition of complex III by antimycin. In the presence of R123, the autofluorescence of NADH was reduced indicating non-radiative energy transfer from NADH to R123. The ratio of the R123 fluorescence signals obtained with the two excitation wavelengths of 355 nm and 488 nm was taken as a measure of mitochondrial NADH. Relative NADH changes were estimated in the presence of the above-mentioned inhibitors. Upon complex I inhibition, mitochondrial NADH was increased by a factor of 1.5. Upon inhibition of complex III, mitochondrial NADH was increased by a factor of 2. We conclude that time-gated spectroscopy combined with non-radiative energy transfer is an appropriate tool for probing mitochondrial enzyme complex deficiencies.

Cellular uptake and efficacy of antisense oligonucleotides against RNAs of two Na(+) channel isoforms.

The effects of 15-mer phosphorothioate antisense oligodeoxynucleotides (aODNs) specifically designed against the RNAs of either of two closely related Na(+) channel isoforms, hSkM1 or hH1, were tested in human myotubes. Fluorescence (3'-fluorescein isothiocyanate) labeling showed that mere incubation of cultures with aODNs did not result in aODN uptake, but liposome-mediated transfer was successful and resulted in cytoplasmic and nuclear localization of ODNs. Intracellular fluorescence was stable for at least 3 days. At 5 microM, the hH1-specific aODN was effective in suppressing ion channel function, but the hSkM1-specific aODN was not. Reverse transcription-polymerase chain reaction gave corresponding results on the mRNA level. However, in HEK-293 cells stably expressing hSkM1, the same hSkM1-specific aODN was able to reduce Na(+) currents (2.4 +/- 0.5 nA, n = 11; controls: 6.5 +/- 1.0 nA, n = 14). We conclude that cellular uptake of aODNs and intracellular access to the RNA target are necessary, but not always sufficient conditions for an effective block of mRNA translation in intact cells.

Anti-GM1 antibodies can block neuronal voltage-gated sodium channels.

Anti-GM1 antibodies, frequently found in the serum of patients with Guillain-Barré syndrome (GBS), have been suggested to interfere with axonal function. We report that IgG anti-GM1 antibodies, raised in rabbits, can reversibly block the voltage-gated Na(+) channels of nerve cells, thus causing a reduction of the excitatory Na(+) current. The block was, however, only substantial when the antibodies were applied together with rabbit complement factors. A solution containing anti-GM1 sera (dilution 1:100) and complement (1:50) reduced the Na(+) current to 0.5 +/- 0.2 times control (mean value +/- SD). Applications of the antibody by itself, complement by itself, or anti-GM2 or anti-GM4 antibodies (1:100) plus complement had little effect. The complexes of anti-GM1 antibodies and complement factors block the ion-conducting pore of the channel directly. In addition, they increase the fraction of channels that are inactivated at the resting potential and alter channel function by changing the membrane surface charge. The described effects may be responsible for conduction slowing and reversible conduction failure in some GBS patients.

An endogenous pentapeptide acting as a sodium channel blocker in inflammatory autoimmune disorders of the central nervous system.

Reversible blockade of sodium channels by endogenous substances has been claimed to account for the fast exacerbations and relapses commonly seen in demyelinating autoimmune diseases. Evidence has been provided that in the cerebrospinal fluid of patients with multiple sclerosis or Guillain-Barré syndrome, a sodium-channel-blocking factor exists that has properties of local anesthetic agents. This factor could contribute to the nerve conduction block and paresis seen in these disorders. We describe here a previously unknown endogenous substance in human cerebrospinal fluid with distinct channel-blocking properties even at very low (0.00001 M) concentrations. The pentapeptide with the sequence Gln-Tyr-Asn-Ala-Asp exerted its blocking action by shifting the steady-state inactivation curve of the sodium channels to more-negative potentials, as most local anesthetics do. In the cerebrospinal fluid of healthy individuals, its concentration was about 3 microM, whereas in patients with multiple sclerosis and Guillain-Barré syndrome, it increased 300-1,400%. At these concentrations, the peptide's blocking efficacy was higher than that of 50 microM lidocaine. At a concentration of 10 microM, lidocaine is able to 'unmask' subclinical lesions in multiple sclerosis; thus, the endogenous pentapeptide may well contribute to the fast changes of symptoms. Furthermore, it may become valuable as a marker of disease activity.

The small sodium-channel blocking factor in the cerebrospinal fluid of multiple sclerosis patients is probably an oligopeptide.

An endogenous factor that is able to reduce the fast transient sodium current of excitable cells has been reported to exist in the cerebrospinal fluid (CSF) of multiple sclerosis (MS) patients. This was confirmed with nine clinically definite MS patients in the acute relapse. In order to purify and chemically identify the factor, microconcentration and gel filtration high-performance liquid chromatography (HPLC) were applied. After each purification step the activity-containing fraction was determined using a biological assay. With all CSFs the activity was contained in the fraction corresponding to 600-800 Da molecular weight, indicating that the factor is chemically homogeneous. The biological activity of the CSF specimens was not correlated to the laboratory CSF data; however, it was correlated to the area under the 210 nm UV light absorption peak in the corresponding chromatogram, i.e. the 600-800 Da MW fraction. As the factor was degradable by acid hydrolysis and a carboxypeptidase, it is suggested that it might be a small peptide.

A small sodium channel blocking factor in the cerebrospinal fluid is preferentially found in Guillain-Barré syndrome: a combined cell physiological and HPLC study.

The cerebrospinal fluid (CSF) of patients with Guillain-Barré syndrome (GBS) contains a low molecular weight factor with sodium channel blocking activity. This study investigated whether such activity also exists in the CSF of patients with other neurological diseases. Further, using high-performance liquid chromatography (HPLC) we tested whether the electrophysiological effect of the CSF is correlated with the size of the corresponding peak in the chromatograms. The existence of sodium channel blocking activity was tested in 27 native CSF samples of three groups of patients (group 1: GBS, n = 13; group 2: other inflammatory diseases, n = 8; group 3: controls, n = 6). NH15-CA2 neuroblastoma x glioma cells in the whole-cell recording configuration was used as a system for assaying the sodium channel blocking activity of CSF specimens. CSF shifted the steady-state inactivation curve of the sodium channels reversibly by -10.2 +/- 4.4 mV in group 1, -6.7 +/- 3.9 mV in group 2, and - 3.5 +/- 2.8 mV in group 3 (P < 0.01). The shift was greater in demyelinating (9.3 +/- 4.7 mV) than in nondemyelinating (5.6 +/- 3.9 mV) diseases (P < 0.04). HPLC analysis of CSFs showed a well separated peak containing the substance responsible for the electrophysiological effect at about 41 min elution time. The peak covered the molecular weight range of 600-800 Da. Sodium channel blocking activity of CSFs and areas of the corresponding peak in the chromatograms were well correlated. We conclude that sodium current inhibition by a low molecular weight factor is generally present but increased in GBS.

A common mutation (epsilon1267delG) in congenital myasthenic patients of Gypsy ethnic origin.

OBJECTIVE: Mutation analysis of the acetylcholine receptor (AChR) epsilon subunit gene in patients with sporadic or autosomal recessive congenital myasthenic syndromes (CMS). BACKGROUND: The nicotinic AChR of skeletal muscle is a neurotransmitter-gated ion channel that mediates synaptic transmission at the vertebrate neuromuscular junction. Mutations in its gene may cause congenital myasthenic syndromes. A recently described mutation in exon 12 of the AChR epsilon subunit (epsilon1267delG) disrupts the cytoplasmic loop and the fourth transmembrane region (M4) of the AChR epsilon subunit. METHODS: Forty-three CMS patients from 35 nonrelated families were clinically classified as sporadic cases of CMS (group III according to European Neuromuscular Centre consensus) and were analyzed for epsilon1267delG by PCR amplification and sequence analysis. RESULTS: The authors report the complete genomic sequence and organization of the gene coding for the epsilon subunit of the human AChR (accession number AF105999). Homozygous epsilon1267delG was identified in 13 CMS patients from 11 independent families. All epsilon1267delG families were of Gypsy or southeastern European origin. Genotype analysis indicated that they derive from a common ancestor (founder) causing CMS in the southeastern European Gypsy population. Phenotype analysis revealed a uniform pattern of clinical features including bilateral ptosis and mild to moderate fatigable weakness of ocular, facial, bulbar, and limb muscles. CONCLUSIONS: The mutation epsilon1267delG might be frequent in European congenital myasthenic syndrome patients of Gypsy ethnic origin. In general, patients (epsilon1267delG) were characterized by the onset of symptoms in early infancy, the presence of ophthalmoparesis, positive response to anticholinesterase treatment, and the benign natural course of the disease.

Mutant channels contribute <50% to Na+ current in paramyotonia congenita muscle.

An important question in the pathophysiology of dominantly inherited diseases, such as channelopathies, is the level of expression of the mutant protein. In our study, we address this issue by comparing the gating defects of two human muscle Na+ channel mutants (R1448C and R1448P) causing paramyotonia congenita in native muscle specimens from two patients with those of the same mutant recombinant channels expressed in human embryonic kidney (HEK-293) cells. Patch-clamp recordings of transfected HEK-293 cells revealed a pronounced slowing of the Na+ current decay, a left-shifted and decreased voltage dependence of steady-state inactivation, and an increased frequency of channel reopenings for mutant compared with wild-type channels. For R1448P channels, inactivation was almost six-fold and for R1448C it was three-fold slower than for wild-type channels. The same defects, though less pronounced, as expected for a disorder with dominant inheritance, were observed for muscle specimens from paramyotonia congenita patients carrying these mutations. Quantitative kinetic analysis of Na+ channel inactivation in the paramyotonic muscle specimens separating wild-type from mutant channels suggested that no more than 38% of the channels in the paramyotonia congenita muscle specimen were of the mutant type. Our data raise the possibility that variability in the ratio of mutant to wild-type Na+ channels in the muscle membrane has an impact on the clinical severity of the phenotype.