New aspects in deriving health-based guidance values for bromate in swimming pool water.

Bromate, classified as a EU CLP 1B carcinogen, is a typical by-product of the disinfection of drinking and swimming pool water. The aim of this study was (a) to provide data on the occurrence of bromate in pool water, (b) to re-evaluate the carcinogenic MOA of bromate in the light of existing data, (c) to assess the possible exposure to bromate via swimming pool water and (d) to inform the derivation of cancer risk-related bromate concentrations in swimming pool water. Measurements from monitoring analysis of 229 samples showed bromate concentrations in seawater pools up to 34 mg/L. A comprehensive non-systematic literature search was done and the quality of the studies on genotoxicity and carcinogenicity was assessed by Klimisch criteria (Klimisch et al., Regul Toxicol Pharmacol 25:1-5, 1997) and SciRAP tool (Beronius et al., J Appl Toxicol, 38:1460-1470, 2018) respectively. Benchmark dose (BMD) modeling was performed using the modeling average mode in BMDS 3.1 and PROAST 66.40, 67 and 69 (human cancer BMDL10; EFSA 2017). For exposure assessment, data from a wide range of sources were evaluated for their reliability. Different target groups (infants/toddlers, children and adults) and exposure scenarios (recreational, sport-active swimmers, top athletes) were considered for oral, inhalation and dermal exposure. Exposure was calculated according to the frequency of swimming events and duration in water. For illustration, cancer risk-related bromate concentrations in pool water were calculated for different target groups, taking into account their exposure using the hBMDL10 and a cancer risk of 1 in 100,000. Convincing evidence was obtained from a multitude of studies that bromate induces oxidative DNA damage and acts as a clastogen in vitro and in vivo. Since statistical modeling of the available genotoxicity data is compatible with both linear as well as non-linear dose-response relationships, bromate should be conservatively considered to be a non-threshold carcinogen. BMD modeling with model averaging for renal cancer studies (Kurokawa et al., J Natl. Cancer Inst, 1983 and 1986a; DeAngelo et al., Toxicol Pathol 26:587-594, 1998) resulted in a median hBMDL10 of 0.65 mg bromate/kg body weight (bw) per day. Evaluation of different age and activity groups revealed that top athletes had the highest exposure, followed by sport-active children, sport-active adults, infants and toddlers, children and adults. The predominant route of exposure was oral (73-98%) by swallowing water, followed by the dermal route (2-27%), while the inhalation route was insignificant (< 0.5%). Accepting the same risk level for all population groups resulted in different guidance values due to the large variation in exposure. For example, for an additional risk of 1 in 100,000, the bromate concentrations would range between 0.011 for top athletes, 0.015 for sport-active children and 2.1 mg/L for adults. In conclusion, the present study shows that health risks due to bromate exposure by swimming pool water cannot be excluded and that large differences in risk exist depending on the individual swimming habits and water concentrations.

Critical evaluation of human health risks due to hydraulic fracturing in natural gas and petroleum production.

The use of hydraulic fracturing (HF) to extract oil and natural gas has increased, along with intensive discussions on the associated risks to human health. Three technical processes should be differentiated when evaluating human health risks, namely (1) drilling of the borehole, (2) hydraulic stimulation, and (3) gas or oil production. During the drilling phase, emissions such as NOx, NMVOCs (non-methane volatile organic compounds) as precursors for tropospheric ozone formation, and SOx have been shown to be higher compared to the subsequent phases. In relation to hydraulic stimulation, the toxicity of frac fluids is of relevance. More than 1100 compounds have been identified as components. A trend is to use fewer, less hazardous and more biodegradable substances; however, the use of hydrocarbons, such as kerosene and diesel, is still allowed in the USA. Methane in drinking water is of low toxicological relevance but may indicate inadequate integrity of the gas well. There is a great concern regarding the contamination of ground- and surface water during the production phase. Water that flows to the surface from oil and gas wells, so-called 'produced water', represents a mixture of flow-back, the injected frac fluid returning to the surface, and the reservoir water present in natural oil and gas deposits. Among numerous hazardous compounds, produced water may contain bromide, arsenic, strontium, mercury, barium, radioactive isotopes and organic compounds, particularly benzene, toluene, ethylbenzene and xylenes (BTEX). The sewage outflow, even from specialized treatment plants, may still contain critical concentrations of barium, strontium and arsenic. Evidence suggests that the quality of groundwater and surface water may be compromised by disposal of produced water. Particularly critical is the use of produced water for watering of agricultural areas, where persistent compounds may accumulate. Air contamination can occur as a result of several HF-associated activities. In addition to BTEX, 20 HF-associated air contaminants are group 1A or 1B carcinogens according to the IARC. In the U.S., oil and gas production (including conventional production) represents the second largest source of anthropogenic methane emissions. High-quality epidemiological studies are required, especially in light of recent observations of an association between childhood leukemia and multiple myeloma in the neighborhood of oil and gas production sites. In conclusion, (1) strong evidence supports the conclusion that frac fluids can lead to local environmental contamination; (2) while changes in the chemical composition of soil, water and air are likely to occur, the increased levels are still often below threshold values for safety; (3) point source pollution due to poor maintenance of wells and pipelines can be monitored and remedied; (4) risk assessment should be based on both hazard and exposure evaluation; (5) while the concentrations of frac fluid chemicals are low, some are known carcinogens; therefore, thorough, well-designed studies are needed to assess the risk to human health with high certainty; (6) HF can represent a health risk via long-lasting contamination of soil and water, when strict safety measures are not rigorously applied.

The German approach to regulate indoor air contaminants.

Indoor air quality (IAQ) and exposure to indoor chemicals are widely discussed in terms of personal discomfort and health risks. In contrast to ambient air and working environments, legally binding regulations are only partially established for indoor contaminants, and other available European guidelines are limited. To correct these deficits, the German Committee on Indoor Guide Values (AIR), formerly known as the Ad hoc Working Group (Ad hoc AG), performed health assessments of indoor air contaminants. The main tasks were to develop toxicologically based indoor air guide values, health-based guideline values, and reference values largely based on the 95th percentile of the concentrations found in a reference population. Here, we provide a comprehensive overview of the indoor air values set in Germany and discuss the basis of their derivation. This overview includes a description of legally binding standards, indoor air guide values for 38 substances or groups, and guidelines for TVOC (total volatile organic compounds), particulate matter, and carbon dioxide as well as risk-related guidelines for carcinogenic substances.

The organotin compounds trimethyltin (TMT) and triethyltin (TET) but not tributyltin (TBT) induce activation of microglia co-cultivated with astrocytes.

The organotin compounds trimethyltin (TMT), triethyltin (TET) and tributyltin (TBT) show different organotoxicities in vivo. While TMT and TET induce a strong neurotoxicity accompanied by microglial and astroglial activation, TBT rather effects the immune system. Previously, we have shown in an in vitro co-culture model that microglial cells can be activated by TMT in the presence of astrocytes. In this study, we wanted to investigate (a) if the neurotoxic organotin compound TET can also activate microglial cells in vitro similar to TMT and (b) if differences between the neurotoxicants TMT and TET on the one side and TBT on the other exist concerning microglial activation. Therefore, purified microglial and astroglial cell cultures from neonatal rat brains were treated either alone or in co-cultures for 24h with different concentrations of TMT, TET or TBT and the basal cytotoxicity and nitric oxide formation was determined. Furthermore, morphological changes of astrocytes were examined. Our results show that microglial activation can be increased in subcytolethal concentrations, but only in the presence of astrocytes and not in microglial cell cultures alone. This increase was induced by the neurotoxicants TMT and TET but not by TBT. Taken together, the differing microglia activating effect of the organotin compounds may contribute to the differing neurotoxic potential of this group of chemicals in vivo. In addition, our results emphasize the need for co-culture systems when studying interactions between different cell types for toxicity assessment.

Time- and activation-dependency of the protective effect of microglia on astrocytes exposed to peroxide-induced oxidative stress.

Microglial as well as astroglial activation (gliosis) are common phenomena of neuropathological processes in the brain. Nevertheless, the role of glial cell communication during gliosis is still not completely understood. The data we present with this work are a part of a bigger study on microglial-astroglial cell communication. The purpose of this study was to examine the impact of microglial-astroglial cell interactions on the cytotoxic effect of hydrogen peroxide (H2O2) on astrocytes and to characterize the microglia-induced astroprotective effect in detail. Therefore, purified cell cultures of microglia and astroglia from neonatal rat brains were used. Astrocytes were pre-treated with conditioned medium from lipopolysaccharides (LPS)-activated microglia and then exposed to oxidative stress induced by H2O2 under varying conditions. Activated microglia significantly reduced the cytotoxic effect of H2O2. As well in the indirect co-culture model using conditioned medium of microglia as in a direct co-culture model with different portions of microglial cells on the astroglial monolayer the astroprotective effect clearly correlated with the degree of microglial activation. The protective effect on astrocytes was induced in less than 24 h and lasted more than 4 days. Taken together, our results indicate that microglial activation might play an important role in the modulation of the oxidative stress resistance of glial tissue during gliosis.

Involvement of benzodiazepine receptors in neuroinflammatory and neurodegenerative diseases: evidence from activated microglial cells in vitro.

Increased binding of a ligand for the peripheral benzodiazepine binding receptor is currently used in PET studies as an in vivo measurement of inflammation in diseases like multiple sclerosis and Alzheimer's disease. Although peripheral-type benzodiazepin receptors (PBRs) are abundant in many cell types and expressed in the CNS physiologically only at low levels, previous reports suggest that after experimental lesions in animal models and in human neurodegenerative/-inflammatory diseases upregulated PBR expression with increased binding of its ligand PK11195 is confined mainly to activated microglia in vivo/in situ. Because the functional role of the PBR is unknown, we confirm by immunohistochemistry and PCR (I) that this receptor is expressed on microglia in vitro and (II) that benzodiazepines modulate proliferation of microglial cells and the release of the inflammatory molecules nitric oxide (NO) and tumor necrosis factor-alpha (TNF-alpha) in cell culture supernatants of primary rat microglia. Compared to lipopolysaccharide-activated controls the release of NO was markedly decreased in cultures treated with benzodiazepines (clonazepam, midazolam, diazepam) and the PBR ligand PK11195. Moreover, release of TNF-alpha and proliferation was significantly inhibited in the benzodiazepine-treated groups. These findings link the in vivo data of elevated PBR levels in neurodegenerative/-inflammatory diseases to a functional role and opens up possible therapeutic intervention targeting the PBR in microglia.

Toxicity of organotin compounds in primary cultures of rat cortical astrocytes.

The neurotoxic organotin compounds trimethyl (TMT) and triethyltin (TET) are known to induce astrogliosis in vivo, which is indicated by an increased synthesis of glial fibrillary acidic protein (GFAP) in astrocytes. In contrast, tributyltin (TBT) does not induce astrogliosis. The aim of this study was to investigate whether trialkyltin derivatives can induce an increased GFAP synthesis in astrocyte cultures in the absence of neurons and whether differences between the action of TMT, TET, and TBT can be detected. Primary cultures of rat cortical astrocytes from 2-day-old rats were grown in 96-well plates until confluency and then exposed to various concentrations of TMT, TET, and TBT for 40 h. Effects on basal cell functions were measured by colorimetric determination of cell protein contents and by assessment of viability by means of the MTT assay. An indirect sandwich ELISA for 96-well plates was used for quantitative measurements of the GFAP content of the cells. All three compounds induced a concentration-dependent cytotoxicity indicated by parallel decreases of protein contents and MTT reduction. Half-maximum cytotoxic concentrations were 3 micromol/L (TBT), 30 micromol/L (TET), and 800 micromol/L (TMT). Cellular GFAP contents were reduced in parallel to cytotoxic action but no increase in GFAP expression at subcytotoxic concentrations could be observed. Thus, the astrocytes were not able to respond to TMT or TET exposure by an increased synthesis of GFAP in the absence of neuronal signals.

Rosetta in CASP4: progress in ab initio protein structure prediction.

Rosetta ab initio protein structure predictions in CASP4 were considerably more consistent and more accurate than previous ab initio structure predictions. Large segments were correctly predicted (>50 residues superimposed within an RMSD of 6.5 A) for 16 of the 21 domains under 300 residues for which models were submitted. Models with the global fold largely correct were produced for several targets with new folds, and for several difficult fold recognition targets, the Rosetta models were more accurate than those produced with traditional fold recognition models. These promising results suggest that Rosetta may soon be able to contribute to the interpretation of genome sequence information.

Effect of Staphylococcus enterotoxin B on the concurrent CD8(+) T cell response to influenza virus infection.

Bacterial superantigens have potent in vivo effects. Respiratory viral infections are often associated with secondary bacterial infections, raising the likelihood of exposure to bacterial superantigens after the initiation of the anti-viral immune response. In this study, the general and V beta-specific effects of exposure to Staphylococcal enterotoxin B (SEB) during influenza virus infection on both the ongoing acute and the subsequent recall CD8(+) T cell responses were analyzed, using the well-characterized murine influenza model system and tetrameric MHC/peptide reagents to directly identify virus-specific T cells. The results show that although superantigen exposure during the primary viral infection caused delayed viral clearance, there was remarkably little effect of SEB on the magnitude or TCR repertoire of the ongoing cytolytic T cell response or on the recall response elicited by secondary viral infection. Thus, despite the well-characterized immunomodulatory effects of SEB, there was surprisingly little interference with concurrent anti-viral immunity.

Alanine is helix-stabilizing in both template-nucleated and standard peptide helices.

Alanine-based peptides of defined sequence and length show measurable helix contents, allowing them to be used as a model system both for analyzing the mechanism of helix formation and for investigating the contributions of side-chain interactions to protein stability. Extensive characterization of many peptide sequences with varying amino acid contents indicates that the favorable helicity of alanine-based peptides can be attributed to the large helix-stabilizing propensity of alanine. Based on their analysis of alanine-rich sequences N-terminally linked to a synthetic helix-inducing template, Kemp and coworkers [Kemp, D. S., Boyd, J. G. & Muendel, C. C. (1991) Nature (London) 352, 451-454; Kemp, D. S., Oslick, S. L. & Allen, T. J. (1996) J. Am. Chem. Soc. 118, 4249-4255] argue that alanine is helix-indifferent, however, and that the favorable helix contents of alanine-based peptides must have some other explanation. Here, we show that the helix contents of template-nucleated sequences are influenced strongly by properties of the template-helix junction. A model in which the helix propensities of residues at the template-peptide junction are treated separately brings the results from alanine-based peptides and template-nucleated helices into agreement. The resulting model provides a physically plausible resolution of the discrepancies between the two systems and allows the helix contents of both template-nucleated and standard peptide helices to be predicted by using a single set of helix propensities. Helix formation in both standard peptides and template-peptide conjugates can be attributed to the large intrinsic helix-forming tendency of alanine.

Solution structure of the sodium channel inactivation gate.

The sodium channel initiates action potentials by opening in response to membrane depolarization. Fast channel inactivation, which is required for proper physiological function, is mediated by a cytoplasmic loop proposed to occlude the ion pore via a hinged lid mechanism with the triad IFM serving as a hydrophobic "latch". The NMR solution structure of the isolated inactivation gate reveals a stably folded core comprised of an alpha-helix capped by an N-terminal turn, supporting a model in which the tightly folded core containing the latch motif pivots on a more flexible hinge region to occlude the pore during inactivation. The structure, in combination with substituted cysteine mutagenesis experiments, indicates that the IFM triad and adjacent Thr are essential components of the latch and suggests differing roles for the residues of the IFMT motif in fast inactivation.

Comparison of NH exchange and circular dichroism as techniques for measuring the parameters of the helix-coil transition in peptides.

Circular dichroism and NH exchange are compared directly as techniques for measuring helix content in peptides and the parameters of the helix-coil transition. To cover a broad range of helix contents, alanine-based peptides with chain lengths varying from 12 to 22 residues are examined over the temperature range from 0.6 to 26.9 degrees C in 1 M sodium chloride, 2H2O. Helix-coil transition theory independently fits both circular dichroism and exchange data, but the helix contents measured by exchange are larger than those measured by circular dichroism. The two techniques are brought into agreement by removing the assumption that the intrinsic chemical exchange rate in the helix is the same as the exchange rate measured for short unstructured model peptides. This modification allows the circular dichroism and NH exchange data to be described by the same set of helix parameters and indicates that the intrinsic exchange rate in the presence of helical structure is reduced approximately 17% relative to the rates measured in unstructured models. To test the possibility that this effect is electrostatic in origin, the sensitivity of the exchange reaction to ionic strength is determined. A substantial dependence of exchange rate on ionic strength is found, but the form of the dependence is complex. In studies of the exchange rates of native proteins, the exchange-competent form of the protein is assumed to exchange with the same rate constant as a blocked dipeptide with the identical amino acid sequences. Our result suggests that this assumption will be seriously in error in some cases because of charge effects in the protein.

Helix propagation and N-cap propensities of the amino acids measured in alanine-based peptides in 40 volume percent trifluoroethanol.

The helix propagation and N-cap propensities of the amino acids have been measured in alanine-based peptides in 40 volume percent trifluoroethanol (40% TFE) to determine if this helix-stabilizing solvent uniformly affects all amino acids. The propensities in 40% TFE are compared with revised values of the helix parameters of alanine-based peptides in water. Revision of the propensities in water is the result of redefining the capping statistical weights and evaluating the helix nucleation constant with N-capping explicitly included in the helix-coil model. The propagation propensities of all amino acids increase in 40% TFE relative to water, but the increases are highly variable. In water, all beta-branched and beta-substituted amino acids are helix breakers. In 40% TFE, the propagation propensities of the nonpolar amino acids increase greatly, leaving charged and neutral polar, beta-substituted amino acids as helix breakers. Glycine and proline are strong helix breakers in both solvents. Free energy differences for helix propagation (delta delta G) between alanine and other nonpolar amino acids are twice as large in water as predicted from side-chain conformational entropies, but delta delta G values in 40% TFE are close to those predicted from side-chain entropies. This dependence of delta delta G on the solvent points to a specific role of water in determining the relative helix propensities of the nonpolar amino acids. The N-cap propensities converge toward a common value in 40% TFE, suggesting that differential solvation by water contributes to the diversity of N-cap values shown by the amino acids.

Models for the 3(10)-helix/coil, pi-helix/coil, and alpha-helix/3(10)-helix/coil transitions in isolated peptides.

Models for the 3(10)-helix/coil and pi-helix/coil equilibria have been derived. The theory is based on classifying residues into helical or nonhelical (coil) conformations. Statistical weights are assigned to residues in a helical conformation with an associated helical hydrogen bond, a helical conformation with no hydrogen bond, an N-cap position, a C-cap position, or the reference coil conformation. The models for alpha-helix formation and 3(10)-helix formation have also been combined to describe a three-state equilibrium in which alpha-helical, 3(10)-helical, and coil conformations are populated. The results are compared with the modified Lifson-Roig theory for the alpha-helix/coil equilibrium. The comparison accounts for the experimental observations that 3(10)-helices tend to be short and pi-helices are not favored for any length. This work may provide a framework for quantitatively rationalizing experimental work on isolated 3(10)-helices and mixed 3(10)-/alpha-helices.

A general two-process model describes the hydrogen exchange behavior of RNase A in unfolding conditions.

When NMR hydrogen exchange was used previously to monitor the kinetics of RNase A unfolding, some peptide NH protons were found to show EX2 exchange (detected by base catalysis) in addition to the expected EX1 exchange, whose rate is limited by the kinetic unfolding process. In earlier work, two groups showed independently that a restricted two-process model successfully fits published hydrogen exchange rates of native RNase A in the range 0-0.7 M guanidinium chloride. We find that this model predicts properties that are very different from the observed properties of the EX2 exchange reactions of RNase A in conditions where guanidine-induced unfolding takes place. The model predicts that EX2 exchange should be too fast to measure by the technique used, whereas it is readily measurable. Possible explanations for the contradiction are considered here, and we show that removing the restriction from the earlier two-process model is sufficient to resolve the contradiction; instead of specifying that exchange caused by global unfolding occurs by the EX2 mechanism, we allow it to occur by the general mechanism, which includes both the EX1 and EX2 cases. It is logical to remove this restriction because global unfolding of RNase A is known to give rise to EX1 exchange in these unfolding conditions. Resolving the contradiction makes it possible to determine whether populated unfolding intermediates contribute to the EX2 exchange, and this question is considered elsewhere. The results and simulations indicate that moderate or high denaturant concentrations readily give rise to EX1 exchange in native proteins. Earlier studies showed that hydrogen exchange in native proteins typically occurs by the EX2 mechanism but that high temperatures or pH values above 7 may give rise to EX1 exchange. High denaturant concentrations should be added to the list of variables likely to cause EX1 exchange.

Addition of side chain interactions to modified Lifson-Roig helix-coil theory: application to energetics of phenylalanine-methionine interactions.

We introduce here i, i + 3 and i, i + 4 side chain interactions into the modified Lifson-Roig helix-coil theory of Doig et al. (1994, Biochemistry 33:3396-3403). The helix/coil equilibrium is a function of initiation, propagation, capping, and side chain interaction parameters. If each of these parameters is known, the helix content of any isolated peptide can be predicted. The model considers every possible conformation of a peptide, is not limited to peptides with only a single helical segment, and has physically meaningful parameters. We apply the theory to measure the i, i + 4 interaction energies between Phe and Met side chains. Peptides with these residues spaced i, i + 4 are significantly more helical than controls where they are spaced i, i + 5. Application of the model yields delta G for the Phe-Met orientation to be -0.75 kcal.mol-1, whereas that for the Met-Phe orientation is -0.54 kcal.mol-1. These orientational preferences can be explained, in part, by rotamer preferences for the interacting side chains. We place Phe-Met i, i + 4 at the N-terminus, the C-terminus, and in the center of the host peptide. The model quantitatively predicts the observed helix contents using a single parameter for the side chain-side chain interaction energy. This result indicates that the model works well even when the interaction is at different locations in the helix.