The Threat Posed by Environmental Contaminants on Neurodevelopment: What Can We Learn from Neural Stem Cells?

Exposure to chemicals may pose a greater risk to vulnerable groups, including pregnant women, fetuses, and children, that may lead to diseases linked to the toxicants' target organs. Among chemical contaminants, methylmercury (MeHg), present in aquatic food, is one of the most harmful to the developing nervous system depending on time and level of exposure. Moreover, certain man-made PFAS, such as PFOS and PFOA, used in commercial and industrial products including liquid repellants for paper, packaging, textile, leather, and carpets, are developmental neurotoxicants. There is vast knowledge about the detrimental neurotoxic effects induced by high levels of exposure to these chemicals. Less is known about the consequences that low-level exposures may have on neurodevelopment, although an increasing number of studies link neurotoxic chemical exposures to neurodevelopmental disorders. Still, the mechanisms of toxicity are not identified. Here we review in vitro mechanistic studies using neural stem cells (NSCs) from rodents and humans to dissect the cellular and molecular processes changed by exposure to environmentally relevant levels of MeHg or PFOS/PFOA. All studies show that even low concentrations dysregulate critical neurodevelopmental steps supporting the idea that neurotoxic chemicals may play a role in the onset of neurodevelopmental disorders.

Neurotoxic mechanisms of triclosan: The antimicrobial agent emerging as a toxicant.

Several scientific studies have suggested a link between increased exposure to pollutants and a rise in the number of neurodegenerative disorders of unknown origin. Notably, triclosan (an antimicrobial agent) is used in concentrations ranging from 0.3% to 1% in various consumer products. Recent studies have also highlighted triclosan as an emerging toxic pollutant due to its increasing global use. However, a definitive link is missing to associate the rising use of triclosan and the growing number of neurodegenerative disorders or neurotoxicity. In this article, we present systematic scientific evidence which are otherwise scattered to suggest that triclosan can indeed induce neurotoxic effects, especially in vertebrate organisms including humans. Mechanistically, triclosan affected important developmental and differentiation genes, structural genes, genes for signaling receptors and genes for neurotransmitter controlling enzymes. Triclosan-induced oxidative stress impacting cellular proteins and homeostasis which triggers apoptosis. Though the scientific evidence collated in this article unequivocally indicates that triclosan can cause neurotoxicity, further epidemiological studies may be needed to confirm the effects on humans.

Dual-targeted magnetic mesoporous silica nanoparticles reduce brain amyloid-ß burden via depolymerization and intestinal metabolism.

Rationale: Active removal of excess peripheral amyloid-ß (Aß) can potentially treat Alzheimer's disease (AD). However, the peripheral clearance of Aß using an anti-Aß monoclonal antibody (mAb) cannot remove PET-detectable Aß within the brain. This may be due to the inability of mAb to cross the blood-brain barrier (BBB) to degrade insoluble brain Aß plaques and block liver dysfunction. Methods: We developed a dual-targeted magnetic mesoporous silica nanoparticle (HA-MMSN-1F12) through surface-coupled Aß42-targeting antibody 1F12 and CD44-targeting ligand hyaluronic acid (HA). Results: HA-MMSN-1F12 had a high binding affinity toward Aß42 oligomers (Kd = 1.27 ± 0.34 nM) and revealed robust degradation of Aß42 aggregates. After intravenous administration of HA-MMSN-1F12 into ten-month-old APP/PS1 mice for three weeks (4 mg/kg/week), HA-MMSN-1F12 could cross the BBB and depolymerize brain Aß plaques into soluble Aß species. In addition, it also avoided hepatic uptake and excreted captured Aß species through intestinal metabolism, thereby reducing brain Aß load and neuroinflammation and improving memory deficits of APP/PS1 mice. Furthermore, the biochemical analysis showed that HA-MMSN-1F12 did not detect any toxic side effects on the liver and kidney. Thus, the efficacy of HA-MMSN-1F12 is associated with the targeted degradation of insoluble brain Aß plaques, avoidance of non-specific hepatic uptake, and excretion of peripheral Aß through intestinal metabolism. Conclusions: The study provides a new avenue for treating brain diseases by excreting disease-causing biohazards using intestinal metabolism.

Are tolerance processes limiting the responses of Hediste diversicolor to cadmium exposure? A multimarker approach.

Cadmium (Cd) is considered a priority hazardous substance under the European Community Directive 2013/39 due to its ecotoxicity. The ragworm Hediste diversicolor (O.F. Müller, 1776), a common species in estuaries and coastal lagoons, plays an important ecological role in these ecosystems and is a suitable bioindicator of environmental chemical contamination. In this study, H. diversicolor was chosen as an ecotoxicological model with the aim of evaluating the responses to Cd contamination, considering a multi-biomarker approach (mortality, biometry, behaviour, Cd bioaccumulation, oxidative stress and damage, and energy metabolism). Also, the hypothesis of different tolerances resulting in different responses was evaluated, by collecting worms from three systems distinctly impacted by metal contamination (Mondego estuary, Óbidos Lagoon and Sado estuary - Portugal). Animals were exposed under laboratory conditions to cadmium (10, 50 and 100 µg/L), for 10 days. Significant differences were observed in responses amongst worms originating from the different sites. Organisms from the less impacted systems revealed greater effects on mortality, biomass decrease and burrowing behaviour, as well as higher bioaccumulation potential, after exposure to Cd. Biochemical and behaviour impairments were observed as a consequence of Cd exposure, although not in a concentration-dependant manner. The results obtained in this study reinforce the importance of integrating endpoint responses, at the individual and sub-individual levels, to assess potential changes induced by pollutants in the physiological status and fitness of H. diversicolor and help to predict what their ecological consequences might be.

Carveol ameliorates mercury-induced oxidative stress, neuroinflammation, and neurodegeneration in a mouse brain.

BACKGROUND: Mercury compounds are the world's third most hazardous substance. Mercury (II) chloride, also known as mercuric chloride (HgCl2), has been shown to have neurotoxic properties in a variety of forms. In numerous investigations, oxidative stress has been established as a key contributor to HgCl2-induced neurotoxicity. Carveol has been researched as an antioxidant and Nrf2-activator in several studies. This study was conducted to investigate if the carveol could protect mice against HgCl2-induced neuronal damage. METHODS: Mice were exposed to a dose of 0.4 mg/kg of HgCl2 and 20 mg/kg of carveol for 21 days. Animals were then subjected to behavioral evaluation through various methods such as open field test (OFT), elevated plus maze test (EPM), morris-water maze test (MWM), and Y-maze test. RESULTS: Results indicated hippocampal-related behavior anomalies which were improved significantly after carveol treatment. Oxidative stress was accompanied by excessive neuroinflammation, which was demonstrated by elevated levels of inflammatory markers such as TNF-α, p-NFkB, and COX-2, and were measured by Western blot, ELISA, and immunohistochemistry. These elevated levels of inflammatory markers were significantly mitigated upon treatment with carveol. To further investigate the participation of the JNK pathway, we used SP-600125 to inhibit JNK, which enhanced the neuroprotective effects of carveol. Moreover, molecular docking and modeling studies were used to validate these effects. CONCLUSION: Our findings indicate that carveol can inhibit the p-JNK pathway, thereby inhibiting HgCl2-induced apoptosis and downregulating the expression of inflammatory mediators.

Toxicant-Induced Metabolic Alterations in Lipid and Amino Acid Pathways Are Predictive of Acute Liver Toxicity in Rats.

Liver disease and disorders associated with aberrant hepatocyte metabolism can be initiated via drug and environmental toxicant exposures. In this study, we tested the hypothesis that gene and metabolic profiling can reveal commonalities in liver response to different toxicants and provide the capability to identify early signatures of acute liver toxicity. We used Sprague Dawley rats and three classical hepatotoxicants: acetaminophen (2 g/kg), bromobenzene (0.4 g/kg), and carbon tetrachloride (0.3 g/kg), to identify early perturbations in liver metabolism after a single acute exposure dose. We measured changes in liver genes and plasma metabolites at two time points (5 and 10 h) and used genome-scale metabolic models to identify commonalities in liver responses across the three toxicants. We found strong correlations for gene and metabolic profiles between the toxicants, indicative of similarities in the liver response to toxicity. We identified several injury-specific pathways in lipid and amino acid metabolism that changed similarly across the three toxicants. Our findings suggest that several plasma metabolites in lipid and amino acid metabolism are strongly associated with the progression of liver toxicity, and as such, could be targeted and clinically assessed for their potential as early predictors of acute liver toxicity.

Developmental exposure of zebrafish to vitamin D receptor acting drugs and environmental toxicants disrupts behavioral function.

Vitamin D receptor (VDR) signaling is important for optimal neurobehavioral development. Disruption of VDR signaling by environmental toxicants during early development might contribute to the etiology of behavioral dysfunction. In the current set of studies, we examined ten compounds known to affect VDR function in vitro for neurobehavioral effects in vivo in zebrafish. Zebrafish embryos were exposed to concentrations of the compounds in their water during the first 5 days post-fertilization. On day 5, the embryos were tested in an alternating light-dark locomotor assay using a computerized video tracking system. We found that most of the compounds produced significant changes in locomotor behavior in exposed zebrafish larvae, although the direction of the effect (i.e., hypo- or hyperactivity) and the sensitivity of the effect to changes in illumination condition varied across the compounds. The nature of the behavioral effects generally corresponded to the effects these compounds have been shown to exert on VDR. These studies lay a foundation for further investigation to determine whether behavioral dysfunction persists into adulthood and if so which behavioral functions are affected. Zebrafish can be useful for screening compounds identified in high throughput in vitro assays to provide an initial test for how those compounds would affect construction and behavioral function of a complex nervous system, helping to bridge the gap between in vitro neurotoxicity assays and mammalian models for risk assessment in humans.

Role of extracellular vesicles in cell-cell communication and inflammation following exposure to pulmonary toxicants.

Extracellular vesicles (EVs) have emerged as key regulators of cell-cell communication during inflammatory responses to lung injury induced by diverse pulmonary toxicants including cigarette smoke, air pollutants, hyperoxia, acids, and endotoxin. Many lung cell types, including epithelial cells and endothelial cells, as well as infiltrating macrophages generate EVs. EVs appear to function by transporting cargo to recipient cells that, in most instances, promote their inflammatory activity. Biologically active cargo transported by EVs include miRNAs, cytokines/chemokines, damage-associated molecular patterns (DAMPs), tissue factor (TF)s, and caspases. Findings that EVs are taken up by target cells such as macrophages, and that this leads to increased proinflammatory functioning provide support for their role in the development of pathologies associated with toxicant exposure. Understanding the nature of EVs responding to toxic exposures and their cargo may lead to the development of novel therapeutic approaches to mitigating lung injury.

In vitro Cytotoxicity and Genotoxicity Analysis of Ten Tannery Chemicals Using SOS/umu Tests and High-content In vitro Micronucleus Tests.

BACKGROUND: More than 2,000 chemicals have been used in the tannery industry. Although some tannery chemicals have been reported to have harmful effects on both human health and the environment, only a few have been subjected to genotoxicity and cytotoxicity evaluations. OBJECTIVE: This study focused on cytotoxicity and genotoxicity of ten tannery chemicals widely used in China. MATERIALS AND METHODS: DNA-damaging effects were measured using the SOS/umu test with Salmonella typhimurium TA1535/pSK1002. Chromosome-damaging and cytotoxic effects were determined with the high-content in vitro Micronucleus test (MN test) using the human-derived cell lines MGC-803 and A549. CONCLUSION: The cytotoxicity of the ten tannery chemicals differed somewhat between the two cell assays, with A549 cells being more sensitive than MGC-803 cells. None of the chemicals induced DNA damage before metabolism, but one was found to have DNA-damaging effects on metabolism. Four of the chemicals, DY64, SB1, DB71 and RR120, were found to have chromosome-damaging effects. A Quantitative Structure-Activity Relationship (QSAR) analysis indicated that one structural feature favouring chemical genotoxicity, Hacceptor-path3-Hacceptor, may contribute to the chromosome-damaging effects of the four MN-test-positive chemicals.

Acute sensitivity of the killifish Nothobranchius furzeri to a combination of temperature and reference toxicants (cadmium, chlorpyrifos and 3,4-dichloroaniline).

Aquatic organisms of inland waters are often subjected to a combination of stressors. Yet, few experiments assess mixed stress effects beyond a select group of standard model organisms. We studied the joint toxicity of reference toxicants and increased temperature on the turquoise killifish, Nothobranchius furzeri, a promising model for ecotoxicological research due to the species' short life cycle and the production of drought-resistant eggs. The acute sensitivity of the larval stage (2dph) to three compounds (cadmium, 3,4-dichloroaniline and chlorpyrifos) was tested in combination with a temperature increase of 4 °C, mimicking global warming. Dose-response relationships were used to calculate 96h-LC50 of 0.28 mg/L (24 °C) and 0.39 mg/L (28 °C) for cadmium, 96h-LC50 of 9.75 mg/L (24 °C) and 6.61 mg/L (28 °C) for 3,4-dichloroaniline and 96h-LC50 of 15.4 µg/L (24 °C) and 14.2 µg/L (28 °C) for chlorpyrifos. After 24 h of exposure, the toxicity of all tested compounds was exacerbated under increased temperature. Furthermore, the interaction effect of cadmium and temperature could be predicted by the stress addition model (SAM). This suggests the applicability of the model for fish and at the same time indicates that the model could be suitable to predict effects of temperature-toxicant interactions.

Using early life stages of marine animals to screen the toxicity of priority hazardous and noxious substances.

This study provides toxicity values for early life stages (ELS) of two phylogenetically distinct marine animal taxa, the sea urchin (Paracentrotus lividus), a deuterostome invertebrate, and the turbot (Scophthalmus maximus), a vertebrate (teleost), when challenged by six hazardous and noxious substances (HNS): aniline, butyl acrylate, m-cresol, cyclohexylbenzene, hexane and trichloroethylene. The aim of the study was to provide preliminary information on toxic effects of representative and relevant priority HNS to assess the risk posed by spills to marine habitats and therefore improve preparedness and the response at the operational level. Selection criteria to include each compound in the study were (1) inclusion in the HASREP (2005) list; (2) presence on the priority list established by Neuparth et al. (2011); (3) paucity of toxicological data (TOXnet and ECOTOX) for marine organisms; (4) behaviour in the water according to the categories defined by the European Behaviour classification system (GESAMP 2002), by selecting compounds with different behaviours in water; and (5) physicochemical and toxicological properties, where available, in order to anticipate the most toxic compounds. Aniline and m-cresol were the most toxic compounds with no observed apical effect concentration (NOAEC) values for sea urchin ranging between 0.01 and 0.1 mg/L, followed by butyl acrylate and cyclohexylbenzene with NOAECs ranging between 0.1 and 1.0 mg/L and trichloroethylene with NOAEC values that were in the range between 1 and 10 mg/L, reflecting their behaviour in water, mostly vapour pressure, but also solubility and log Kow. Hexane was toxic only for turbot embryos, due to its neurotoxic effects, and not for sea urchin larvae, at concentrations in the range between 1 and 10 mg/L. The concentrations tested were of the same order of magnitude for both species, and it was observed that sea urchin embryos (length of the longest arm) are more sensitive than turbot eggs larvae (hatching and cumulative mortality rates) to the HNS tested (except hexane). For this specific compound, concentrations up to 70 mg/L were tested in sea urchin larvae and no effects were observed on the length of the larvae. Both tests were found to be complementary depending on behaviour in water and toxicity target of the compounds analysed.

The effect of the feeding pattern of complex industrial wastewater on activated sludge characteristics and the chemical and ecotoxicological effluent quality.

Research has demonstrated that the feeding pattern of synthetic wastewater plays an important role in sludge characteristics during biological wastewater treatment. Although considerable research has been devoted to synthetic wastewater, less attention has been paid to industrial wastewater. In this research, three different feeding strategies were applied during the treatment of tank truck cleaning (TTC) water. This industry produces highly variable wastewaters that are often loaded with hazardous chemicals, which makes them challenging to treat with activated sludge (AS). In this study, it is shown that the feeding pattern has a significant influence on the settling characteristics. Pulse feeding resulted in AS with a sludge volume index (SVI) of 68 ± 15 mL gMLSS-1. Slowly and continuously fed AS had to contend with unstable SVI values that fluctuated between 100 and 600 mL gMLSS-1. These fluctuations were clearly caused by the feeding solution. The obtained settling characteristics are being supported by the microscopic analysis, which revealed a clear floc structure for the pulse fed AS. Ecotoxicological effluent assessment with bacteria, Crustacea and algae identified algae as the most sensitive organism for all effluents from all different reactors. Variable algae growth inhibitions were measured between the different reactors. The chemical and ecotoxicological effluent quality was comparable between the reactors.

Cytogenetic Biomonitoring in Buccal Mucosal Cells from Municipal Solid Waste Collectors.

Waste collectors collect, transport, and process the garbage produced by people living in the city. Nowadays, this activity requires special attention due to the environmental impact of garbage and its potential consequences on human health. The aim of this study was to evaluate potential cytotoxic and mutagenic effects of garbage collection on waste collectors. For this purpose, a total of 47 male waste collectors aged from 24 to 53 years were included in the experimental group. A total of 30 men matched by age were used as the control group. Cytotoxicity and mutagenicity were analyzed by micronucleus test in buccal mucosaI cells. No statistically significant difference (p>0.05) in the frequency of micronuclei was detected in the waste collectors when compared to controls. Nevertheless, higher frequencies of karyolysis and pyknosis (p<0.05) were detected in buccal mucosaI cells from waste collectors when compared to matched controls. Taken together, our results indicate that waste collectors comprise an at-risk group as a result of increased cytotoxicity apparent from buccal mucosa cells.

Comparative study of preparation of hazardous drugs with different closed-system drug transfer devices by means of simulation with fluorescein / Estudio comparativo de preparación de fármacos peligrosos con varias modalidades de sistemas cerrados mediante simulación con fluoresceína

Objectives: The level of environmental contamination generated during preparation and administration of hazardous drugs using different valve closed-systems and their combinations was compared. The actual impact on the overall time of preparation of cytostatics and the economic cost of the different modalities were also compared. Methods: Comparative study of the preparation of fluorescein mixtures with different modalities of valve closed-system combinations. Environmental contamination was detected in critical points of connection, and in splashes produced at any other points. The main variable was qualitative detection of contamination by splashes through ultraviolet light when modalities with or without a connector were compared. A final number of 160 mixtures were prepared to detect differences of at least 5%. Results: Splashes were produced in 7 preparations without a connector (p = 0.015). No significant differences (p = 0.445) were detected either in the use of a supporting vial spike vs an anchoring spike, or in the ChemoCLAVE® system vs valve systems with Fleboflex® solutions. Contamination at any critical point was produced in all preparations. The use of a supporting vial spike, syringe connector and bag solution with Luer connection was the most efficient modality. Conclusions: A syringe connector is needed to guarantee a closed system. Anchoring spikes do not show higher advantages as compared with supporting vial spikes. Fleboflex® solutions with Luer bags are more efficient than ChemoCLAVE® and show similar safety. However, connections of these closed systems are not leak-tight, and it is therefore important to continue studies of contamination of the different closed system transfer devices (AU)

Objetivo: Comparar la contaminación generada durante la elaboración y administración de fármacos peligrosos con diferentes componentes de sistemas cerrados y de manera secundaria, seleccionar el sistema más eficiente. Material y métodos: Estudio comparativo de elaboración de mezclas de fluoresceína con diferentes combinaciones de sistemas cerrados de tipo valvular. Se consideró contaminación ambiental la detectada en los puntos críticos de conexión y las salpicaduras generadas en cualquier otro punto distinto. La variable principal fue la detección cualitativa mediante luz ultravioleta de contaminación por salpicaduras al comparar las modalidades con y sin conector. Se calculó un tamaño muestral de 160 preparaciones por modalidad, para detectar diferencias de al menos un 5%. Resultados: Se produjeron salpicaduras en 7 preparaciones, todas sin conector (p = 0,015). No se encontraron diferencias entre utilizar punzón de apoyo o de anclaje (p = 0,445), ni entre el sistema ChemoCLAVE® vs sistema valvular con sueros Fleboflex®. En todas las preparaciones se produjo contaminación en algún punto crítico. La utilización de punzones de apoyo, conectores y sueros luer se ha identificado como la modalidad más eficiente. Conclusiones: Es importante utilizar el conector de jeringa para que el sistema sea completamente cerrado. El uso de punzones de anclaje no parece presentar ventajas frente a los de apoyo y la combinación con los sueros Fleboflex® presenta una seguridad similar al sistema ChemoCLAVE®. Sin embargo, las conexiones de estos sistemas no son secas y, por tanto, es importante continuar con estudios de contaminación que comparen diferentes sistema (AU)

Part 1. Statistical Learning Methods for the Effects of Multiple Air Pollution Constituents.

INTRODUCTION: The United States Environmental Protection Agency (U.S. EPA*) currently regulates individual air pollutants on a pollutant-by-pollutant basis, adjusted for other pollutants and potential confounders. However, the National Academies of Science concluded that a multipollutant regulatory approach that takes into account the joint effects of multiple constituents is likely to be more protective of human health. Unfortunately, the large majority of existing research had focused on health effects of air pollution for one pollutant or for one pollutant with control for the independent effects of a small number of copollutants. Limitations in existing statistical methods are at least partially responsible for this lack of information on joint effects. The goal of this project was to fill this gap by developing flexible statistical methods to estimate the joint effects of multiple pollutants, while allowing for potential nonlinear or nonadditive associations between a given pollutant and the health outcome of interest. METHODS: We proposed Bayesian kernel machine regression (BKMR) methods as a way to simultaneously achieve the multifaceted goals of variable selection, flexible estimation of the exposure-response relationship, and inference on the strength of the association between individual pollutants and health outcomes in a health effects analysis of mixtures. We first developed a BKMR variable-selection approach, which we call component-wise variable selection, to make estimating such a potentially complex exposure-response function possible by effectively using two types of penalization (or regularization) of the multivariate exposure-response surface. Next we developed an extension of this first variable-selection approach that incorporates knowledge about how pollutants might group together, such as multiple constituents of particulate matter that might represent a common pollution source category. This second grouped, or hierarchical, variable-selection procedure is applicable when groups of highly correlated pollutants are being studied. To investigate the properties of the proposed methods, we conducted three simulation studies designed to evaluate the ability of BKMR to estimate environmental mixtures responsible for health effects under potentially complex but plausible exposure-response relationships. An attractive feature of our simulation studies is that we used actual exposure data rather than simulated values. This real-data simulation approach allowed us to evaluate the performance of BKMR and several other models under realistic joint distributions of multipollutant exposure. The simulation studies compared the two proposed variable-selection approaches (component-wise and hierarchical variable selection) with each other and with existing frequentist treatments of kernel machine regression (KMR). After the simulation studies, we applied the newly developed methods to an epidemiologic data set and to a toxicologic data set. To illustrate the applicability of the proposed methods to human epidemiologic data, we estimated associations between short-term exposures to fine particulate matter constituents and blood pressure in the Maintenance of Balance, Independent Living, Intellect, and Zest in the Elderly (MOBILIZE) Boston study, a prospective cohort study of elderly subjects. To illustrate the applicability of these methods to animal toxicologic studies, we analyzed data on the associations between both blood pressure and heart rate in canines exposed to a composition of concentrated ambient particles (CAPs) in a study conducted at the Harvard T. H. Chan School of Public Health (the Harvard Chan School; formerly Harvard School of Public Health; Bartoli et al. 2009). RESULTS: We successfully developed the theory and computational tools required to apply the proposed methods to the motivating data sets. Collectively, the three simulation studies showed that component-wise variable selection can identify important pollutants within a mixture as long as the correlations among pollutant concentrations are low to moderate. The hierarchical variable-selection method was more effective in high-dimension, high-correlation settings. Variable selection in existing frequentist KMR models can incur inflated type I error rates, particularly when pollutants are highly correlated. The analyses of the MOBILIZE data yielded evidence of a linear and additive association of black carbon (BC) or Cu exposure with standing diastolic blood pressure (DBP), and a linear association of S exposure with standing systolic blood pressure (SBP). Cu is thought to be a marker of urban road dust associated with traffic; and S is a marker of power plant emissions or regional long-range transported air pollution or both. Therefore, these analyses of the MOBILIZE data set suggest that emissions from these three source categories were most strongly associated with hemodynamic responses in this cohort. In contrast, in the Harvard Chan School canine study, after controlling for an overall effect of CAPs exposure, we did not observe any associations between DBP or SBP and any elemental concentrations. Instead, we observed strong evidence of an association between Mn concentrations and heart rate in that heart rate increased linearly with increasing concentrations of Mn. According to the positive matrix factorization (PMF) source apportionment analyses of the multipollutant data set from the Harvard Chan School Boston Supersite, Mn loads on the two factors that represent the mobile and road dust source categories. The results of the BKMR analyses in both the MOBILIZE and canine studies were similar to those from existing linear mixed model analyses of the same multipollutant data because the effects have linear and additive forms that could also have been detected using standard methods. CONCLUSIONS: This work provides several contributions to the KMR literature. First, to our knowledge this is the first time KMR methods have been used to estimate the health effects of multipollutant mixtures. Second, we developed a novel hierarchical variable-selection approach within BKMR that is able to account for the structure of the mixture and systematically handle highly correlated exposures. The analyses of the epidemiologic and toxicologic data on associations between fine particulate matter constituents and blood pressure or heart rate demonstrated associations with constituents that are typically associated with traffic emissions, power plants, and long-range transported pollutants. The simulation studies showed that the BKMR methods proposed here work well for small to moderate data sets; more work is needed to develop computationally fast methods for large data sets. This will be a goal of future work.

Part 2. Development of Enhanced Statistical Methods for Assessing Health Effects Associated with an Unknown Number of Major Sources of Multiple Air Pollutants.

A major difficulty with assessing source-specific health effects is that source-specific exposures cannot be measured directly; rather, they need to be estimated by a source-apportionment method such as multivariate receptor modeling. The uncertainty in source apportionment (uncertainty in source-specific exposure estimates and model uncertainty due to the unknown number of sources and identifiability conditions) has been largely ignored in previous studies. Also, spatial dependence of multipollutant data collected from multiple monitoring sites has not yet been incorporated into multivariate receptor modeling. The objectives of this project are (1) to develop a multipollutant approach that incorporates both sources of uncertainty in source-apportionment into the assessment of source-specific health effects and (2) to develop enhanced multivariate receptor models that can account for spatial correlations in the multipollutant data collected from multiple sites. We employed a Bayesian hierarchical modeling framework consisting of multivariate receptor models, health-effects models, and a hierarchical model on latent source contributions. For the health model, we focused on the time-series design in this project. Each combination of number of sources and identifiability conditions (additional constraints on model parameters) defines a different model. We built a set of plausible models with extensive exploratory data analyses and with information from previous studies, and then computed posterior model probability to estimate model uncertainty. Parameter estimation and model uncertainty estimation were implemented simultaneously by Markov chain Monte Carlo (MCMC*) methods. We validated the methods using simulated data. We illustrated the methods using PM2.5 (particulate matter ≤ 2.5 µm in aerodynamic diameter) speciation data and mortality data from Phoenix, Arizona, and Houston, Texas. The Phoenix data included counts of cardiovascular deaths and daily PM2.5 speciation data from 1995-1997. The Houston data included respiratory mortality data and 24-hour PM2.5 speciation data sampled every six days from a region near the Houston Ship Channel in years 2002-2005. We also developed a Bayesian spatial multivariate receptor modeling approach that, while simultaneously dealing with the unknown number of sources and identifiability conditions, incorporated spatial correlations in the multipollutant data collected from multiple sites into the estimation of source profiles and contributions based on the discrete process convolution model for multivariate spatial processes. This new modeling approach was applied to 24-hour ambient air concentrations of 17 volatile organic compounds (VOCs) measured at nine monitoring sites in Harris County, Texas, during years 2000 to 2005. Simulation results indicated that our methods were accurate in identifying the true model and estimated parameters were close to the true values. The results from our methods agreed in general with previous studies on the source apportionment of the Phoenix data in terms of estimated source profiles and contributions. However, we had a greater number of statistically insignificant findings, which was likely a natural consequence of incorporating uncertainty in the estimated source contributions into the health-effects parameter estimation. For the Houston data, a model with five sources (that seemed to be Sulfate-Rich Secondary Aerosol, Motor Vehicles, Industrial Combustion, Soil/Crustal Matter, and Sea Salt) showed the highest posterior model probability among the candidate models considered when fitted simultaneously to the PM2.5 and mortality data. There was a statistically significant positive association between respiratory mortality and same-day PM2.5 concentrations attributed to one of the sources (probably industrial combustion). The Bayesian spatial multivariate receptor modeling approach applied to the VOC data led to a highest posterior model probability for a model with five sources (that seemed to be refinery, petrochemical production, gasoline evaporation, natural gas, and vehicular exhaust) among several candidate models, with the number of sources varying between three and seven and with different identifiability conditions. Our multipollutant approach assessing source-specific health effects is more advantageous than a single-pollutant approach in that it can estimate total health effects from multiple pollutants and can also identify emission sources that are responsible for adverse health effects. Our Bayesian approach can incorporate not only uncertainty in the estimated source contributions, but also model uncertainty that has not been addressed in previous studies on assessing source-specific health effects. The new Bayesian spatial multivariate receptor modeling approach enables predictions of source contributions at unmonitored sites, minimizing exposure misclassification and providing improved exposure estimates along with their uncertainty estimates, as well as accounting for uncertainty in the number of sources and identifiability conditions.

Grouping of histone deacetylase inhibitors and other toxicants disturbing neural crest migration by transcriptional profiling.

Functional assays, such as the "migration inhibition of neural crest cells" (MINC) developmental toxicity test, can identify toxicants without requiring knowledge on their mode of action (MoA). Here, we were interested, whether (i) inhibition of migration by structurally diverse toxicants resulted in a unified signature of transcriptional changes; (ii) whether statistically-identified transcript patterns would inform on compound grouping even though individual genes were little regulated, and (iii) whether analysis of a small group of biologically-relevant transcripts would allow the grouping of compounds according to their MoA. We analyzed transcripts of 35 'migration genes' after treatment with 16 migration-inhibiting toxicants. Clustering, principal component analysis and correlation analyses of the data showed that mechanistically related compounds (e.g. histone deacetylase inhibitors (HDACi), PCBs) triggered similar transcriptional changes, but groups of structurally diverse toxicants largely differed in their transcriptional effects. Linear discriminant analysis (LDA) confirmed the specific clustering of HDACi across multiple separate experiments. Similarity of the signatures of the HDACi trichostatin A and suberoylanilide hydroxamic acid to the one of valproic acid (VPA), suggested that the latter compound acts as HDACi when impairing neural crest migration. In conclusion, the data suggest that (i) a given functional effect (e.g. inhibition of migration) can be associated with highly diverse signatures of transcript changes; (ii) statistically significant grouping of mechanistically-related compounds can be achieved on the basis of few genes with small regulations. Thus, incorporation of mechanistic markers in functional in vitro tests may support read-across procedures, also for structurally un-related compounds.

Decomposition of birch leaves in heavily polluted industrial barrens: relative importance of leaf quality and site of exposure.

The decrease in litter decomposition rate in polluted habitats is well documented, but the factors that explain the observed variation in the magnitude of this pollution effect on litter decomposition remain poorly understood. We explored effects of environmental conditions and leaf quality on decomposition rate of mountain birch (Betula pubescens ssp. czerepanovii) leaves in a heavily polluted industrial barren near the nickel-copper smelter at Monchegorsk. Litter bags filled with leaves collected from two heavily polluted barren sites and from two control forest sites were buried at 2.5-cm depth and exposed for 2 and 4 years at each of these four sites. The relative mass loss of native leaves in the industrial barren during 2 years of exposure was reduced to 49% of the loss observed in the unpolluted forest. We found a similar reduction in mass loss when leaves from control sites were exposed to polluted sites and when leaves from polluted sites were exposed to control sites. We conclude that the reduction in leaf litter decomposition in an industrial barren is caused by pollution-induced changes in both environmental conditions and leaf quality. This reduction is much smaller than expected, given the four-fold decrease in soil microbial activity and nearly complete extinction of saprophagous invertebrates in the polluted soil. We suggest that a longer snowless period and higher spring and summer temperatures at the barren sites have partially counterbalanced the adverse effects caused by the toxicity of metal pollutants.

Characteristics of chemosensory disorders--results from a survey.

This survey was undertaken to investigate the nature of chemosensory dysfunction in relation to the underlying cause, severity, and course of the disease and to elucidate their clinical significance. A total of 269 patients (116 men and 149 women) with chemosensory disorders participated in the survey. Approximately 89 % had olfactory loss, either alone or in combination with taste loss and 2 % had gustatory loss alone; 7.4 % had chemosensory distortions. Dysosmia was significantly higher in those on more than four medicines per day (p < 0.02). Most patients (51.6 %) reported sudden onset of symptoms. Self-reported etiologies included: flu/infection (39.4 %), medication intake (13 %), sinusitis (12 %), operation (10.7 %), head-trauma (9.3 %), and not-specified (12.7 %). The most frequent complaint was diminished pleasure from eating followed by a decrease in general quality of life (QoL). Patients with gradual onset of symptoms or long-standing disease complained the least (p < 0.005). Of all etiologies, patients with SND complained the most (p < 0.04). Overall, 18.6 % ate more and 7.3 % ate less, 7.5 % changed their food preferences, and 19 % reported weight gain and 15.8 % weight loss. Haptic feedback was considered more important than visual appeal and acoustic feedback of food. Older patients however valued visual appeal more. When asked directly, 63 % reported having experienced household-mishaps, 58.1 % problems with social communication, 56.8 % reported having changed their sexual behavior and 35.9 % suffered depression. 60.4 % did not cope well with the changes in their lives. Women reported more problems than men, particularly relating to interpersonal communication (64.5 vs. 57.6 %) and mood (47.9 vs. 40.9 %). Women also had more frequent spontaneous recollections of smells (p < 0.02). Chemosensory disorders have a significant impact on QoL. Reduced pleasure from eating is the predominant complaint of patients seeking medical attention. The steeper the onset of symptoms, the worse the ability to cope with changes in daily life. Older patients value the visual appeal, whereas younger patients value the haptic feedback of foods.

Development of a convenient in vivo hepatotoxin assay using a transgenic zebrafish line with liver-specific DsRed expression.

Previously we have developed a transgenic zebrafish line (LiPan) with liver-specific red fluorescent protein (DsRed) expression under the fabp10a promoter. Since red fluorescence in the liver greatly facilitates the observation of liver in live LiPan fry, we envision that the LiPan zebrafish may provide a useful tool in analyses of hepatotoxicity based on changes of liver red fluorescence intensity and size. In this study, we first tested four well-established hepatotoxins (acetaminophen, aspirin, isoniazid and phenylbutazone) in LiPan fry and demonstrated that these hepatotoxins could significantly reduce both liver red fluorescence and liver size in a dosage-dependent manner, thus the two measurable parameters could be used as indicators of hepatotoxicity. We then tested the LiPan fry with nine other chemicals including environmental toxicants and human drugs. Three (mefenamic acid, lindane, and arsenate) behave like hepatotoxins in reduction of liver red fluorescence, while three others (17ß-estradiol, TCDD [2,3,7,8-tetrachlorodibenzo-p-dioxin] and NDMA [N-nitrosodimethylamine]) caused increase of liver red fluorescence and the liver size. Ethanol and two other chemicals, amoxicillin (antibiotics) and chlorphenamine (pain killer) did not resulted in significant changes of liver red fluorescence and liver size. By quantitative RT-PCR analysis, we found that the changes of red fluorescence intensity caused by different chemicals correlated to the changes of endogenous fabp10a RNA expression, indicating that the measured hepatotoxicity was related to fatty acid transportation and metabolism. Finally we tested a mixture of four hepatotoxins and observed a significant reduction of red fluorescence in the liver at concentrations below the lowest effective concentrations of individual hepatotoxins, suggesting that the transgenic zebrafish assay is capable of reporting compound hepatotoxicity effect from chemical mixtures. Thus, the LiPan transgenic fry provide a rapid and convenient in vivo hepatotoxicity assay that should be applicable to high-throughput hepatotoxicity test in drug screening as well as in biomonitoring environmental toxicants.