Prenatal exposure to polycyclic aromatic hydrocarbons and executive functions at school age: Results from a combined cohort study.

BACKGROUND: Executive functions develop rapidly in childhood, enabling problem-solving, focused attention, and planning. Exposures to environmental toxicants in pregnancy may impair healthy executive function development in children. There is increasing concern regarding polycyclic aromatic hydrocarbons (PAHs) given their ability to transfer across the placenta and the fetal blood-brain barrier, yet evidence from epidemiological studies is limited. METHODS: We examined associations between prenatal PAH exposure and executive functions in 814 children of non-smoking mothers from two U.S. cohorts in the ECHO-PATHWAYS Consortium. Seven mono-hydroxylated PAH metabolites were measured in mid-pregnancy urine and analyzed individually and as mixtures. Three executive function domains were measured at age 8-9: cognitive flexibility, working memory, and inhibitory control. A composite score quantifying overall performance was further calculated. We fitted linear regressions adjusted for socio-demographics, maternal health behaviors, and psychological measures, and examined modification by child sex and stressful life events in pregnancy. Bayesian kernel machine regression was performed to estimate the interactive and overall effects of the PAH mixture. RESULTS: The results from primary analysis of linear regressions were generally null, and no modification by child sex or maternal stress was indicated. Mixture analyses suggested several pairwise interactions between individual PAH metabolites in varied directions on working memory, particularly interactions between 2/3/9-FLUO and other PAH metabolites, but no overall or individual effects were evident. CONCLUSION: We conducted a novel exploration of PAH-executive functions association in a large, combined sample from two cohorts. Although findings were predominantly null, the study carries important implications for future research and contributes to evolving science regarding developmental origins of diseases.

Sex-specific associations between placental corticotropin releasing hormone and problem behaviors in childhood.

Placental corticotropin-releasing hormone (pCRH) is a neuroactive peptide produced in high concentrations in mid-late pregnancy, during key periods of fetal brain development. Some evidence suggests that higher pCRH exposure during gestation is associated with adverse neurodevelopment, particularly in female offspring. In 858 mother-child dyads from the sociodemographically diverse CANDLE cohort (Memphis, TN), we examined: (1) the slope of pCRH rise in mid-late pregnancy and (2) estimated pCRH at delivery as a measure of cumulative prenatal exposure. When children were 4 years-old, mothers reported on problem behaviors using the Child Behavior Checklist (CBCL) and cognitive performance was assessed by trained psychologists using the Stanford-Binet Intelligence Scales. We fitted linear regression models examining pCRH in relation to behavioral and cognitive performance measures, adjusting for covariates. Using interaction models, we evaluated whether associations differed by fetal sex, breastfeeding, and postnatal neighborhood opportunity. In the full cohort, log-transformed pCRH measures were not associated with outcomes; however, we observed sex differences in some models (interaction p-values≤0.01). In male offspring, an interquartile (IQR) increase in pCRH slope (but not estimated pCRH at delivery), was positively associated with raw Total (ß=3.06, 95%CI: 0.40, 5.72), Internalizing (ß=0.89, 95%CI: 0.03, 1.76), and Externalizing (ß=1.25, 95%CI: 0.27, 2.22) Problem scores, whereas, in females, all associations were negative (Total Problems: ß=-1.99, 95%CI: -3.89, -0.09; Internalizing: ß=-0.82, 95%CI: -1.42, -0.23; Externalizing: ß=-0.56, 95%CI: -1.34, 0.22). No associations with cognitive performance were observed nor did we observe moderation by breastfeeding or postnatal neighborhood opportunity. Our results provide further evidence that prenatal pCRH exposure may impact subsequent child behavior in sex-specific ways, however in contrast to prior studies suggesting adverse impacts in females, steeper mid-gestation pCRH rise was associated with more problem behaviors in males, but fewer in females.

A dynamic in vitro developing testis model reflects structures and functions of testicular development in vivo.

To better define appropriate applications of our 3-dimensional testicular co-culture as a model for reproductive toxicology, we evaluated the ability of the model to capture structural and functional elements that can be targeted by reproductive toxicants. Testicular co-cultures were prepared from postnatal day 5 male rats and cultured with a Matrigel overlay. Following a 2-day acclimation period, we characterized functional pathway dynamics by evaluating morphology, protein expression, testosterone concentrations, and global gene expression at a range of timepoints from experimental days 0-21. Western blotting confirmed expression of Sertoli cell, Leydig cell, and spermatogonial cell-specific protein markers. Testosterone detected in cell culture media indicates active testosterone production. Quantitative pathway analysis identified Gene Ontology biological processes enriched among genes significantly changing over the course of 21 days. Processes enriched among genes significantly increasing through time include general developmental processes (morphogenesis, tissue remodeling, etc.), steroid regulation, Sertoli cell development, immune response, and stress and apoptosis. Processes enriched among genes significantly decreasing over time include several related to male reproductive development (seminiferous tubule development, male gonad development, Leydig cell differentiation, Sertoli cell differentiation), all of which appear to peak in expression between days 1 and 5 before decreasing at later timepoints. This analysis provides a temporal roadmap for specific biological process of interest for reproductive toxicology in the model and anchors the model to sensitive phases of in vivo development, helping to define the relevance of the model for in vivo processes.

Alteration of oral microbiome composition in children living with pesticide-exposed farm workers.

Our prior work shows that azinphos-methyl pesticide exposure is associated with altered oral microbiomes in exposed farmworkers. Here we extend this analysis to show the same association pattern is also evident in their children. Oral buccal swab samples were analyzed at two time points, the apple thinning season in spring-summer 2005 for 78 children and 101 adults and the non-spray season in winter 2006 for 62 children and 82 adults. The pesticide exposure for the children were defined by the farmworker occupation of the cohabitating household adult and the blood azinphos-methyl detection of the cohabitating adult. Oral buccal swab 16S rRNA sequencing determined taxonomic microbiota proportional composition from concurrent samples from both adults and children. Analysis of the identified bacteria showed significant proportional changes for 12 of 23 common oral microbiome genera in association with azinphos-methyl detection and farmworker occupation. The most common significantly altered genera had reductions in the abundance of Streptococcus, suggesting an anti-microbial effect of the pesticide. Principal component analysis of the microbiome identified two primary clusters, with association of principal component 1 to azinphos-methyl blood detection and farmworker occupational status of the household. The children's buccal microbiota composition clustered with their household adult in ∼95% of the households. Household adult farmworker occupation and household pesticide exposure is associated with significant alterations in their children's oral microbiome composition. This suggests that parental occupational exposure and pesticide take-home exposure pathways elicit alteration of their children's microbiomes.

Prenatal polycyclic aromatic hydrocarbon (PAH) exposure in relation to placental corticotropin releasing hormone (pCRH) in the CANDLE pregnancy cohort.

Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous endocrine-disrupting combustion by-products that have been linked to preterm birth. One possible mechanism is through disruption of placental corticotropin releasing hormone (pCRH), a key hormone implicated in parturition. As an extension of recent research identifying pCRH as a potential target of endocrine disruption, we examined maternal PAH exposure in relation to pCRH in a large, diverse sample. Participants, drawn from the CANDLE cohort, part of the ECHO-PATHWAYS Consortium, completed study visits at 16-29 weeks (V1) and 22-39 weeks (V2) gestation (n=812). Seven urinary mono-hydroxylated PAH metabolites (OH-PAHs) were measured at V1 and serum pCRH at V1 and V2. Associations between individual log-transformed OH-PAHs (as well as two summed PAH measures) and log(pCRH) concentrations across visits were estimated using mixed effects models. Minimally-adjusted models included gestational age and urinary specific gravity, while fully-adjusted models also included sociodemographic characteristics. We additionally evaluated effect modification by pregnancy complications, fetal sex, and maternal childhood trauma history. We observed associations between 2-OH-Phenanthrene (2-OH-PHEN) and rate of pCRH change that persisted in fully adjusted models (ß=0.0009, 0.00006, 0.0017), however, positive associations with other metabolites (most notably 3-OH-Phenanthrene and 1-Hydroxypyrene) were attenuated after adjustment for sociodemographic characteristics. Associations tended to be stronger at V1 compared to V2 and we observed no evidence of effect modification by pregnancy complications, fetal sex, or maternal childhood trauma history. In conclusion, we observed modest evidence of association between OH-PAHs, most notably 2-OH-PHEN, and pCRH in this sample. Additional research using serial measures of PAH exposure is warranted, as is investigation of alternative mechanisms that may link PAHs and timing of birth, such as inflammatory, epigenetic, or oxidative stress pathways.

Maternal exposure to urinary polycyclic aromatic hydrocarbons (PAH) in pregnancy and childhood asthma in a pooled multi-cohort study.

BACKGROUND: Prenatal exposure to polycyclic aromatic hydrocarbons (PAH) may increase risk of pediatric asthma, but existing human studies are limited. OBJECTIVES: We estimated associations between gestational PAHs and pediatric asthma in a diverse US sample and evaluated effect modification by child sex, maternal asthma, and prenatal vitamin D status. METHODS: We pooled two prospective pregnancy cohorts in the ECHO PATHWAYS Consortium, CANDLE and TIDES, for an analytic sample of N = 1296 mother-child dyads. Mono-hydroxylated PAH metabolites (OH-PAHs) were measured in mid-pregnancy urine. Mothers completed the International Study on Allergies and Asthma in Childhood survey at child age 4-6 years. Poisson regression with robust standard errors was used to estimate relative risk of current wheeze, current asthma, ever asthma, and strict asthma associated with each metabolite, adjusted for potential confounders. We used interaction models to assess effect modification. We explored associations between OH-PAH mixtures and outcomes using logistic weighted quantile sum regression augmented by a permutation test to control Type 1 errors. RESULTS: The sociodemographically diverse sample spanned five cities. Mean (SD) child age at assessment was 4.4 (0.4) years. While there was little evidence that either individual OH-PAHs or mixtures were associated with outcomes, we observed effect modification by child sex for most pairs of OH-PAHs and outcomes, with adverse associations specific to females. For example, a 2-fold increase in 2-hydroxy-phenanthrene was associated with current asthma in females but not males (RRfemale = 1.29 [95 % CI: 1.09, 1.52], RRmale = 0.95 [95 % CI: 0.79, 1.13]; pinteraction = 0.004). There was no consistent evidence of modification by vitamin D status or maternal asthma. DISCUSSION: This analysis, the largest cohort study of gestational PAH exposure and childhood asthma to date, suggests adverse associations for females only. These preliminary findings are consistent with hypothesized endocrine disruption properties of PAHs, which may lead to sexually dimorphic effects.

The effects of gene × environment interactions on silver nanoparticle toxicity in the respiratory system: An adverse outcome pathway.

The Adverse Outcome Pathway (AOP) framework is serving as a basis to integrate new data streams in order to enhance the power of predictive toxicology. AOP development for engineered nanomaterials (ENM), including silver nanoparticles (AgNP), is currently lagging behind other chemicals of regulatory interest due to our limited understanding of the mechanism by which underlying genetics or diseases directly modify host response to AgNP exposures. This also highlights the importance of considering the Aggregate Exposure Pathway (AEP) framework, which precedes the AOP framework and outlines source to target site exposure. The AEP and AOP frameworks interface at the target site, where a molecular initiating event (MIE) occurs and is followed by key events (KE) for adverse cellular and organ responses along a biological pathway and ends with the adverse organism response. The primary goal of this study is to use AgNP to interrogate the AEP-AOP framework by organizing and integrating in vitro dose-response data and in vivo exposure-response data from previous studies to evaluate the effects of interactions between host genetic and acquired factors, or gene × environment interactions (G × E), on AgNP toxicity in the respiratory system. Using this framework will help us to identify plausible key event relationships (KER) between MIE and adverse organism responses when KE are not measured using the same assay in order to derive future predictive models, guide research, and support development of tools for making risk-based, regulatory decisions on ENM. This article is categorized under: Toxicology and Regulatory Issues in Nanomedicine > Toxicology of Nanomaterials Toxicology and Regulatory Issues in Nanomedicine > Regulatory and Policy Issues in Nanomedicine.

The effects of genotype × phenotype interactions on silver nanoparticle toxicity in organotypic cultures of murine tracheal epithelial cells.

Silver nanoparticles (AgNP) are used in multiple applications but primarily in the manufacturing of antimicrobial products. Previous studies have identified AgNP toxicity in airway epithelial cells, but no in vitro studies to date have used organotypic cultures as a high-content in vitro model of the conducting airway to characterize the effects of interactions between host genetic and acquired factors, or gene × phenotype interactions (G × P), on AgNP toxicity. In the present study, we derived organotypic cultures from primary murine tracheal epithelial cells (MTEC) to characterize nominal and dosimetric dose-response relationships for AgNPs with a gold core on barrier dysfunction, glutathione (GSH) depletion, reactive oxygen species (ROS) production, lipid peroxidation, and cytotoxicity across two genotypes (A/J and C57BL/6J mice), two phenotypes ('Normal' and 'Type 2 [T2]-Skewed'), and two exposures (an acute exposure of 24 h and a subacute exposure of 4 h, every other day, over 5 days [5 × 4 h]). We characterized the 'T2-Skewed' phenotype as an in vitro model of chronic respiratory diseases, which was marked by increased sensitivity to AgNP-induced barrier dysfunction, GSH depletion, ROS production, lipid peroxidation, and cytotoxicity, suggesting that asthmatics are a sensitive population to AgNP exposures in occupational settings. This also suggests that exposure limits, which should be based upon the most sensitive population, should be derived using in vitro and in vivo models of chronic respiratory diseases. This study highlights the importance of considering dosimetry as well as G × P effects when screening and prioritizing potential respiratory toxicants. Such in vitro studies can be used to inform regulatory policy aimed at special protections for all populations.

Characterizing the Neurodevelopmental Pesticide Exposome in a Children's Agricultural Cohort.

The exposome provides a conceptual model for identifying and characterizing lifetime environmental exposures and resultant health effects. In this study, we applied key exposome concepts to look specifically at the neurodevelopmental pesticide exposome, which focuses on exposures to pesticides that have the potential to cause an adverse neurodevelopmental impact. Using household dust samples from a children's agricultural cohort located in the Yakima Valley of Washington state, we identified 87 individual pesticides using liquid chromatography-tandem mass spectrometry. A total of 47 of these have evidence of neurotoxicity included in the Environmental Protection Agency (EPA) (re)registration materials. We used a mixed effects model to model trends in pesticide exposure. Over the two study years (2005 and 2011), we demonstrate a significant decrease in the neurodevelopmental pesticide exposome across the cohort, but particularly among farmworker households. Additional analysis with a non-parametric binomial analysis that weighted the levels of potentially neurotoxic pesticides detected in household dust by their reference doses revealed that the decrease in potentially neurotoxic pesticides was largely a result of decreases in some of the most potent neurotoxicants. Overall, this study provides evidence that the neurodevelopmental pesticide exposome framework is a useful tool in assessing the effectiveness of specific interventions in reducing exposure as well as setting priorities for future targeted actions.

The Effects of Genotype × Phenotype Interactions on Transcriptional Response to Silver Nanoparticle Toxicity in Organotypic Cultures of Murine Tracheal Epithelial Cells.

The airway epithelium is critical for maintaining innate and adaptive immune responses, and occupational exposures that disrupt its immune homeostasis may initiate and amplify airway inflammation. In our previous study, we demonstrated that silver nanoparticles (AgNP), which are engineered nanomaterials used in multiple applications but primarily in the manufacturing of many antimicrobial products, induce toxicity in organotypic cultures derived from murine tracheal epithelial cells (MTEC), and those differentiated toward a "Type 2 [T2]-Skewed" phenotype experienced an increased sensitivity to AgNP toxicity, suggesting that asthmatics could be a sensitive population to AgNP exposures in occupational settings. However, the mechanistic basis for this genotype × phenotype (G × P) interaction has yet to be defined. In this study, we conducted transcriptional profiling using RNA-sequencing to predict the enrichment of specific canonical pathways and upstream transcriptional regulators to assist in defining a mechanistic basis for G × P effects on AgNP toxicity. Organotypic cultures were derived from MTEC across 2 genetically inbred mouse strains (A/J and C57BL/6J mice), 2 phenotypes ("Normal" and "T2-Skewed"), and 1 AgNP exposure (an acute 24 h exposure) to characterize G × P effects on transcriptional response to AgNP toxicity. The "T2-Skewed" phenotype was marked by increased pro-inflammatory T17 responses to AgNP toxicity, which are significant predictors of neutrophilic/difficult-to-control asthma and suggests that asthmatics could be a sensitive population to AgNP exposures in occupational settings. This study highlights the importance of considering G × P effects when identifying these sensitive populations, whose underlying genetics or diseases could directly modify their response to AgNP exposures.

Anchoring a dynamic in vitro model of human neuronal differentiation to key processes of early brain development in vivo.

We characterize temporal pathway dynamics of differentiation in an in vitro neurotoxicity model with the aim of informing design and interpretation of toxicological assays. Human neural progenitor cells (hNPCs) were cultured in differentiation conditions up to 21 days. Genes significantly changed through time were identified and grouped according to temporal dynamics. Quantitative pathway analysis identified gene ontology (GO) terms enriched among significantly changed genes and provided a temporal roadmap of pathway trends in vitro. Gene expression in hNPCs was compared with publicly available gene expression data from developing human brain tissue in vivo. Quantitative pathway analysis of significantly changed genes and targeted analysis of specific pathways of interest identified concordance between in vivo and in vitro expression associated with proliferation, migration, differentiation, synapse formation, and neurotransmission. Our analysis anchors gene expression patterns in vitro to sensitive windows of in vivo development, helping to define appropriate applications of the model.

Longitudinal, Seasonal, and Occupational Trends of Multiple Pesticides in House Dust.

BACKGROUND: Children are especially vulnerable to pesticide exposure and can suffer lasting health effects. Because children of farmworkers are exposed to a variety of pesticides throughout development, it is important to explore temporal patterns of coexposures. OBJECTIVES: The objectives of this study were to characterize the pesticide co-exposures, determine how they change over time, and assess differences between farmworker and nonfarmworker households. METHODS: Dust collected from 40 farmworker and 35 nonfarmworker households in the Yakima Valley of the State of Washington in 2005 and then again in 2011 was analyzed for 99 pesticides. Eighty-seven pesticides representing over 28 classes were detected. Pesticides were grouped into classes using U.S. EPA pesticide chemical classifications, and trends in concentrations were analyzed at the class level. RESULTS: Levels of organophosphates, pyridazinones, and phenols significantly decreased between 2005 and 2011 in both farmworker and nonfarmworker households. Levels of anilides, 2,6-dinitroanilines, chlorophenols, triclosan, and guanidines significantly increased in both farmworker and nonfarmworker households in 2011 vs. 2005. Among farmworkers alone, there were significantly lower levels of N-methyl carbamates and neonicotinoids in 2011. CONCLUSIONS: We observed significant reductions in the concentrations of many pesticides over time in both farmworker and nonfarmworker households. Although nonfarmworker households generally had lower concentrations of pesticides, it is important to note that in comparison with NHANES participants, nonfarmworkers and their families still had significantly higher concentrations of urinary pesticide metabolites. This finding highlights the importance of detailed longitudinal exposure monitoring to capture changes in agricultural and residential pesticide use over time. This foundation provides an avenue to track longitudinal pesticide exposures in an intervention or regulatory context. https://doi.org/10.1289/EHP3644.

Application of improved approach to evaluate a community intervention to reduce exposure of young children living in farmworker households to organophosphate pesticides.

The take-home pathway is a significant source of organophosphate pesticide exposure for young children (3-5 years old) living with an adult farmworker. This avoidable exposure pathway is an important target for intervention. We selected 24 agricultural communities in the Yakima Valley of Washington State and randomly assigned them to receive an educational intervention (n = 12) to reduce children's pesticide exposure or usual care (n = 12). We assessed exposure to pesticides in nearly 200 adults and children during the pre and post-intervention periods by measuring metabolites in urine. We compared pre- and post-intervention exposures by expressing the child's pesticide metabolite concentration as a fraction of the adult's concentration living in the same household, because the amount of pesticides applied during the collection periods varied. Exposures in our community were consistently higher, sometimes above the 95th percentile of the exposures reported by the National Health and Nutrition Examination Survey (NHANES). While intervention and control communities demonstrated a reduction in the ratio of child to adult exposure, this reduction was more pronounced in intervention communities (2.7-fold, p < 0.001 compared to 1.7-fold, p = 0.052 for intervention and control, respectively). By examining the child/adult biomarker ratio, we demonstrated that our community-based intervention was effective in reducing pesticide exposure to children in agricultural communities.

Evaluation of the relationship between residential orchard density and dimethyl organophosphate pesticide residues in house dust.

Reducing residential pesticide exposure requires identification of exposure pathways. Compared to the agriculture worker 'take-home' and residential use pathways, evidence of the 'drift' pathway to pesticide exposure has been inconsistent. Questionnaire data from individuals (n = 99) and dust samples (n = 418) from households across three growing seasons in 2011 were from the For Healthy Kids! study. Summed dimethyl organophosphate pesticide (OP) (Azinphos-Methyl, Phosmet, and Malathion) concentrations were quantified from house dust samples. Spatially-weighted orchard densities surrounding households were calculated based on various distances from homes. Regression models tested associations between orchard density, residential pesticide use, agriculture worker residents, and summed dimethyl OP house dust concentrations. Estimated relationships between orchard density and dimethyl OP in house dust were mixed: a 5% increase in orchard density resulted in 0.3 and 0.5% decreases in dimethyl OP house dust concentrations when considering land-cover 750 m or 1250 m away from households, respectively, but null associations with land-cover 60 m or 200 m away. Dimethyl OP house dust concentrations were 400% higher within homes where at least two residents were agriculture workers. Despite inconclusive evidence for the drift pathway due to potential for bias, relationships between number of agriculture workers and dimethyl OP house dust concentration underscores the take-home pathway.

Blood Biochemical and Hematological Study after Subacute Intravenous Injection of Gold and Silver Nanoparticles and Coadministered Gold and Silver Nanoparticles of Similar Sizes.

BACKGROUND: To investigate the effect of subacute intravenous administration AgNP (silver nanoparticles, 10 nm) and AuNP (gold nanoparticles, 12.8 nm) and AgNP/AuNP mixture to blood biochemistry, hematology, and platelet coagulation, subacute toxicity study was conducted. METHODS: AuNP and AgNP in which their size distribution was not statistically different, mixed or separate, were injected into the caudal vein of male Sprague-Dawley rats for 4 weeks. The rats were allowed to recover for a further 4 weeks in order to examine systemic toxicity expressed in the blood biochemistry and hematology. The dose groups (5 males per group for the administration and 3 males for the recovery) consisted of 7 divisions, i.e., control, AgNP (with a low dose of 10 µg/kg/day and a high dose of 100 µg/kg/day), AuNP (with a low dose of 10 µg/kg/day and a high dose of 100 µg/kg/day), and mixed AgNP/AuNP (with a low dose of 10/10 µg/kg/day and a high dose of 100/100 µg/kg/day). RESULTS: There were no significant dose-related changes in the hematology and blood biochemical values for the rats. Coagulation time in terms of the active partial thromboplastin time (APTT) and prothrombin time (PT) did not show any significant changes, when compared to the control group. CONCLUSION: The subacute injection of AuNP and AgNP or their mixture did not induce any noticeable systemic toxicity.

Using primary organotypic mouse midbrain cultures to examine developmental neurotoxicity of silver nanoparticles across two genetic strains.

Micromass culture systems have been developed as three-dimensional organotypic in vitro alternatives to test developmental toxicity. We have optimized a murine-based embryonic midbrain micromass system in two genetic strains to evaluate neurodevelopmental effects of gold-cored silver nanoparticles (AgNPs) of differing sizes and coatings-20 nm AgCitrate, 110 nm AgCitrate, and 110 nm AgPVP. AgNPs are increasingly used in consumer, commercial, and medical products for their antimicrobial properties and observations of Ag in adult and fetal brain following in vivo exposures to AgNPs have led to concerns about the potential for AgNPs to elicit adverse effects on neurodevelopment and neurological function. Cytotoxicity was assessed at three time points of development by both nominal dose and by dosimetric dose. Ag dosimetry was assessed in cultures and the gold core component of the AgNPs was used as a tracer for determination of uptake of intact AgNPs and silver dissolution from particles in the culture system. Results by both nominal and dosimetric dose show cell death increased significantly in a dose-dependent manner at later time points (days 15 and 22 in vitro) that coincide with differentiation stages of development in both strains. When assessed by dosimetric dose, cultures were more sensitive to smaller particles, despite less uptake of Ag in smaller particles in both strains.

Variability in metagenomic samples from the Puget Sound: Relationship to temporal and anthropogenic impacts.

Whole-metagenome sequencing (WMS) has emerged as a powerful tool to assess potential public health risks in marine environments by measuring changes in microbial community structure and function in uncultured bacteria. In addition to monitoring public health risks such as antibiotic resistance determinants, it is essential to measure predictors of microbial variation in order to identify natural versus anthropogenic factors as well as to evaluate reproducibility of metagenomic measurements.This study expands our previous metagenomic characterization of Puget Sound by sampling new nearshore environments including the Duwamish River, an EPA superfund site, and the Hood Canal, an area characterized by highly variable oxygen levels. We also resampled a wastewater treatment plant, nearshore and open ocean sites introducing a longitudinal component measuring seasonal and locational variations and establishing metagenomics sampling reproducibility. Microbial composition from samples collected in the open sound were highly similar within the same season and location across different years, while nearshore samples revealed multi-fold seasonal variation in microbial composition and diversity. Comparisons with recently sequenced predominant marine bacterial genomes helped provide much greater species level taxonomic detail compared to our previous study. Antibiotic resistance determinants and pollution and detoxification indicators largely grouped by location showing minor seasonal differences. Metal resistance, oxidative stress and detoxification systems showed no increase in samples proximal to an EPA superfund site indicating a lack of ecosystem adaptation to anthropogenic impacts. Taxonomic analysis of common sewage influent families showed a surprising similarity between wastewater treatment plant and open sound samples suggesting a low-level but pervasive sewage influent signature in Puget Sound surface waters. Our study shows reproducibility of metagenomic data sampling in multiple Puget Sound locations while establishing baseline measurements of antibiotic resistance determinants, pollution and detoxification systems. Combining seasonal and longitudinal data across these locations provides a foundation for evaluating variation in future studies.

Short-term inhalation study of graphene oxide nanoplates.

Graphene oxides possess unique physicochemical properties with important potential applications in electronics, pharmaceuticals, and medicine. However, the toxicity following inhalation exposure to graphene oxide has not yet been clarified. Therefore, this study conducted a short-term graphene oxide inhalation toxicity analysis using a nose-only inhalation exposure system and male Sprague-Dawley rats. A total of four groups (15 rats per group) were exposed: (1) control (fresh air), (2) low concentration (0.76 ± 0.16 mg/m3), (3) moderate concentration (2.60 ± 0.19 mg/m3), and (4) high concentration (9.78 ± 0.29 mg/m3). The rats were exposed to graphene oxide for 6 h/day for 5 days, followed by recovery for 1, 3, and 21 days. No significant body or organ weight changes were noted after the short-term exposure or during the recovery period. Similarly, no significant systemic effects of toxicological importance were noted in the hematological assays, bronchoalveolar lavage fluid (BAL) inflammatory markers, BAL fluid cytokines, or blood biochemical assays following the graphene oxide exposure or during the post-exposure observation period. Moreover, no significant differences were observed in the BAL cell differentials, such as lymphocytes, macrophages, or polymorphonuclear cells. Graphene oxide-ingested alveolar macrophages as a spontaneous clearance reaction were observed in the lungs of all the concentration groups from post 1 day to post 21 days. Histopathological examination of the liver and kidneys did not reveal any significant test-article-relevant histopathological lesions. Importantly, similar to previously reported graphene inhalation data, this short-term nose-only inhalation study found only minimal or unnoticeable graphene oxide toxicity in the lungs and other organs.

In vitro to in vivo benchmark dose comparisons to inform risk assessment of quantum dot nanomaterials.

Engineered nanomaterials are currently under review for their potential toxicity; however, their use in consumer/commercial products has continued to outpace risk assessments. In vitro methods may be utilized as tools to improve the efficiency of risk assessment approaches. We propose a framework to compare relationships between previously published in vitro and in vivo toxicity assessments of cadmium-selenium containing quantum dots (QDs) using benchmark dose (BMD) and dosimetric assessment methods. Although data were limited this approach was useful for identifying sensitive assays and strains. In vitro studies assessed effects of QDs in three pulmonary cell types across two mouse strains. Significant dose-response effects were modeled and a standardized method of BMD analysis was performed as a function of both exposure dose and dosimetric dose. In vivo studies assessed pulmonary effects of QD exposure across eight mouse strains. BMD analysis served as a basis for relative comparison with in vitro studies. We found consistent responses in common endpoints between in vitro and in vivo studies. Strain sensitivity was consistent between in vitro and in vivo studies, showing A/J mice more sensitive to QDs. Cell types were found to differentially take up QDs. Dosimetric adjustments identified similar sensitivity among cell types. Thus, BMD analysis can be used as an effective tool to compare the sensitivity of different strains, cell types, and assays to QDs. These methods allow for in vitro assays to be used to predict in vivo responses, improve the efficiency of in vivo studies, and allow for prioritization of nanomaterial assessments. This article is categorized under: Toxicology and Regulatory Issues in Nanomedicine > Toxicology of Nanomaterials Toxicology and Regulatory Issues in Nanomedicine > Regulatory and Policy Issues in Nanomedicine.

Characterization of 3D embryonic C57BL/6 and A/J mouse midbrain micromass in vitro culture systems for developmental neurotoxicity testing.

In vitro micromass culture systems have been proposed as an alternative method for developmental toxicity assessment to reduce the need for resource-intensive in vivo toxicity testing. In this study, a three-dimensional in vitro embryonic mouse midbrain culture system is characterized in two mouse strains to facilitate gene x environment considerations. Gestational day (GD) 11 C57BL/6 or GD 12 A/J mouse midbrain cells were isolated and cultured in high-density micromass format for 22days in vitro (DIV). Hematoxylin intensity and protein content revealed that neuronal differentiation increases linearly over time in both C57BL/6 and A/J cultures. Protein expression showed time-dependent proliferation markers (PCNA) increased significantly between DIV 4-6 compared to DIV 1. Early and late differentiation markers (e.g. ß-tubulin III and NMDAɛ1) were expressed between DIV 6-8 and DIV 8-15, respectively. Immunohistochemistry and protein expression results for proliferation and differentiation markers were concordant. Protein expression patterns for the two mouse strain micromass systems were similar. This study characterizes a novel method for investigating early neurogenesis and may be used to characterize neurodevelopmental toxicity in vitro. Our findings show how the use of different mouse strains in neurodevelopmental studies may extend test systems for gene and environment interaction studies.