Screening persistent organic pollutants for effects on testosterone and estrogen synthesis at human-relevant concentrations using H295R cells in 96-well plates.

Many persistent organic pollutants (POPs) are suspected endocrine disruptors and it is important to investigate their effects at low concentrations relevant to human exposure. Here, the OECD test guideline #456 steroidogenesis assay was downscaled to a 96-well microplate format to screen 24 POPs for their effects on viability, and testosterone and estradiol synthesis using the human adrenocortical cell line H295R. The compounds (six polyfluoroalkyl substances, five organochlorine pesticides, ten polychlorinated biphenyls and three polybrominated diphenyl ethers) were tested at human-relevant levels (1 nM to 10 µM). Increased estradiol synthesis, above the OECD guideline threshold of 1.5-fold solvent control, was shown after exposure to 10 µM PCB-156 (153%) and PCB-180 (196%). Interestingly, the base hormone synthesis varied depending on the cell batch. An alternative data analysis using a linear mixed-effects model that include multiple independent experiments and considers batch-dependent variation was therefore applied. This approach revealed small but statistically significant effects on estradiol or testosterone synthesis for 17 compounds. Increased testosterone levels were demonstrated even at 1 nM for PCB-74 (18%), PCB-99 (29%), PCB-118 (16%), PCB-138 (19%), PCB-180 (22%), and PBDE-153 (21%). The MTT assay revealed significant effects on cell viability after exposure to 1 nM of perfluoroundecanoic acid (12%), 3 nM PBDE-153 (9%), and 10 µM of PCB-156 (6%). This shows that some POPs can interfere with endocrine signaling at concentrations found in human blood, highlighting the need for further investigation into the toxicological mechanisms of POPs and their mixtures at low concentrations relevant to human exposure.

Evaluation of Nontargeted Mass Spectral Data Acquisition Strategies for Water Analysis and Toxicity-Based Feature Prioritization by MS2Tox.

The machine-learning tool MS2Tox can prioritize hazardous nontargeted molecular features in environmental waters, by predicting acute fish lethality of unknown molecules based on their MS2 spectra, prior to structural annotation. It has yet to be investigated how the extent of molecular coverage, MS2 spectra quality, and toxicity prediction confidence depend on sample complexity and MS2 data acquisition strategies. We compared two common nontargeted MS2 acquisition strategies with liquid chromatography high-resolution mass spectrometry for structural annotation accuracy by SIRIUS+CSI:FingerID and MS2Tox toxicity prediction of 191 reference chemicals spiked to LC-MS water, groundwater, surface water, and wastewater. Data-dependent acquisition (DDA) resulted in higher rates (19-62%) of correct structural annotations among reference chemicals in all matrices except wastewaters, compared to data-independent acquisition (DIA, 19-50%). However, DIA resulted in higher MS2 detection rates (59-84% DIA, 37-82% DDA), leading to higher true positive rates for spectral library matching, 40-73% compared to 34-72%. DDA resulted in higher MS2Tox toxicity prediction accuracy than DIA, with root-mean-square errors of 0.62 and 0.71 log-mM, respectively. Given the importance of MS2 spectral quality, we introduce a "CombinedConfidence" score to convey relative confidence in MS2Tox predictions and apply this approach to prioritize potentially ecotoxic nontargeted features in environmental waters.

Chemical Exposomics in Human Plasma by Lipid Removal and Large-Volume Injection Gas Chromatography-High-Resolution Mass Spectrometry.

For comprehensive chemical exposomics in blood, analytical workflows are evolving through advances in sample preparation and instrumental methods. We hypothesized that gas chromatography-high-resolution mass spectrometry (GC-HRMS) workflows could be enhanced by minimizing lipid coextractives, thereby enabling larger injection volumes and lower matrix interference for improved target sensitivity and nontarget molecular discovery. A simple protocol was developed for small plasma volumes (100-200 µL) by using isohexane (H) to extract supernatants of acetonitrile-plasma (A-P). The HA-P method was quantitative for a wide range of hydrophobic multiclass target analytes (i.e., log Kow > 3.0), and the extracts were free of major lipids, thereby enabling robust large-volume injections (LVIs; 25 µL) in long sequences (60-70 h, 70-80 injections) to a GC-Orbitrap HRMS. Without lipid removal, LVI was counterproductive because method sensitivity suffered from the abundant matrix signal, resulting in low ion injection times to the Orbitrap. The median method quantification limit was 0.09 ng/mL (range 0.005-4.83 ng/mL), and good accuracy was shown for a certified reference serum. Applying the method to plasma from a Swedish cohort (n = 32; 100 µL), 51 of 103 target analytes were detected. Simultaneous nontarget analysis resulted in 112 structural annotations (12.8% annotation rate), and Level 1 identification was achieved for 7 of 8 substances in follow-up confirmations. The HA-P method is potentially scalable for application in cohort studies and is also compatible with many liquid-chromatography-based exposomics workflows.

Longitudinal Exposomics in a Multiomic Wellness Cohort Reveals Distinctive and Dynamic Environmental Chemical Mixtures in Blood.

Chemical exposomes can now be comprehensively measured in human blood, but knowledge of their variability and longitudinal stability is required for robust application in cohort studies. Here, we applied high-resolution chemical exposomics to plasma of 46 adults, each sampled 6 times over 2 years in a multiomic cohort, resulting in 276 individual exposomes. In addition to quantitative analysis of 83 priority target analytes, we discovered and semiquantified substances that have rarely or never been reported in humans, including personal care products, pesticide transformation products, and polymer additives. Hierarchical cluster analysis for 519 confidently annotated substances revealed unique and distinctive coexposures, including clustered pesticides, poly(ethylene glycols), chlorinated phenols, or natural substances from tea and coffee; interactive heatmaps were publicly deposited to support open exploration of the complex (meta)data. Intraclass correlation coefficients (ICC) for all annotated substances demonstrated the relatively low stability of the exposome compared to that of proteome, microbiome, and endogenous small molecules. Implications are that the chemical exposome must be measured more frequently than other omics in longitudinal studies and four longitudinal exposure types are defined that can be considered in study design. In this small cohort, mixed-effect models nevertheless revealed significant associations between testosterone and perfluoroalkyl substances, demonstrating great potential for longitudinal exposomics in precision health research.

High-Resolution Mass Spectrometry for Human Exposomics: Expanding Chemical Space Coverage.

In the modern "omics" era, measurement of the human exposome is a critical missing link between genetic drivers and disease outcomes. High-resolution mass spectrometry (HRMS), routinely used in proteomics and metabolomics, has emerged as a leading technology to broadly profile chemical exposure agents and related biomolecules for accurate mass measurement, high sensitivity, rapid data acquisition, and increased resolution of chemical space. Non-targeted approaches are increasingly accessible, supporting a shift from conventional hypothesis-driven, quantitation-centric targeted analyses toward data-driven, hypothesis-generating chemical exposome-wide profiling. However, HRMS-based exposomics encounters unique challenges. New analytical and computational infrastructures are needed to expand the analysis coverage through streamlined, scalable, and harmonized workflows and data pipelines that permit longitudinal chemical exposome tracking, retrospective validation, and multi-omics integration for meaningful health-oriented inferences. In this article, we survey the literature on state-of-the-art HRMS-based technologies, review current analytical workflows and informatic pipelines, and provide an up-to-date reference on exposomic approaches for chemists, toxicologists, epidemiologists, care providers, and stakeholders in health sciences and medicine. We propose efforts to benchmark fit-for-purpose platforms for expanding coverage of chemical space, including gas/liquid chromatography-HRMS (GC-HRMS and LC-HRMS), and discuss opportunities, challenges, and strategies to advance the burgeoning field of the exposome.

Maternal pre-pregnancy BMI influences the associations between bisphenol and phthalate exposures and maternal weight changes and fat accumulation.

BACKGROUND: Bisphenols and phthalates are two classes of endocrine-disrupting chemicals (EDCs) thought to influence weight and adiposity. Limited research has investigated their influence on maternal weight changes, and no prior work has examined maternal fat mass. We examined the associations between exposure to these chemicals during pregnancy and multiple maternal weight and fat mass outcomes. METHODS: This study included a sample of 318 women enrolled in a Canadian prospective pregnancy cohort. Second trimester urinary concentrations of 2 bisphenols and 12 phthalate metabolites were quantified. Self-reported and measured maternal weights and measured skinfold thicknesses were used to calculate gestational weight gain, 3-months and 3- to 5-years postpartum weight retention, late pregnancy fat mass gain, total postpartum fat mass loss, and late postpartum fat mass retention. Adjusted robust regressions examined associations between chemicals and outcomes in the entire study population and sub-groups stratified by pre-pregnancy body mass index (BMI). Bayesian kernel machine regression examined chemical mixture effects. RESULTS: Among women with underweight or normal pre-pregnancy BMIs, MBzP was negatively associated with weight retention at 3- to 5-years postpartum (B = -0.04, 95%CI: -0.07, -0.01). Among women with overweight or obese pre-pregnancy BMIs, MEHP and MMP were positively associated with weight retention at 3-months and 3- to 5-years postpartum, respectively (B's = 0.12 to 0.63, 95%CIs: 0.02, 1.07). DEHP metabolites and MCNP were positively associated with late pregnancy fat mass gain and late postpartum fat mass retention (B's = 0.04 to 0.18, 95%CIs: 0.001, 0.32). Further, the mixture of EDCs was positively associated with late pregnancy fat mass gain. CONCLUSION: In this cohort, pre-pregnancy BMI was a key determinant of the associations between second trimester exposure to bisphenols and phthalates and maternal weight changes and fat accumulation. Investigations of underlying physiological mechanisms, windows of susceptibility, and impacts on maternal and infant health are needed.

Phospholipid Removal for Enhanced Chemical Exposomics in Human Plasma.

The challenge of chemical exposomics in human plasma is the 1000-fold concentration gap between endogenous substances and environmental pollutants. Phospholipids are the major endogenous small molecules in plasma, thus we validated a chemical exposomics protocol with an optimized phospholipid-removal step prior to targeted and non-targeted liquid chromatography high-resolution mass spectrometry. Increased injection volume with negligible matrix effect permitted sensitive multiclass targeted analysis of 77 priority analytes; median MLOQ = 0.05 ng/mL for 200 µL plasma. In non-targeted acquisition, mean total signal intensities of non-phospholipids were enhanced 6-fold in positive (max 28-fold) and 4-fold in negative mode (max 58-fold) compared to a control method without phospholipid removal. Moreover, 109 and 28% more non-phospholipid molecular features were detected by exposomics in positive and negative mode, respectively, allowing new substances to be annotated that were non-detectable without phospholipid removal. In individual adult plasma (100 µL, n = 34), 28 analytes were detected and quantified among 10 chemical classes, and quantitation of per- and polyfluoroalkyl substances (PFAS) was externally validated by independent targeted analysis. Retrospective discovery and semi-quantification of PFAS-precursors was demonstrated, and widespread fenuron exposure is reported in plasma for the first time. The new exposomics method is complementary to metabolomics protocols, relies on open science resources, and can be scaled to support large studies of the exposome.

Nontarget Analysis of Polluted Surface Waters in Bangladesh Using Open Science Workflows.

Nontarget mass spectrometry has great potential to reveal patterns of water contamination globally through community science, but few studies are conducted in low-income countries, nor with open-source workflows, and few datasets are FAIR (Findable, Accessible, Interoperable, Reusable). Water was collected from urban and rural rivers around Dhaka, Bangladesh, and analyzed by liquid chromatography high-resolution mass spectrometry in four ionization modes (electrospray ionization ±, atmospheric pressure chemical ionization ±) with data-independent MS2 acquisition. The acquisition strategy was complementary: 19,427 and 7365 features were unique to ESI and APCI, respectively. The complexity of water pollution was revealed by >26,000 unique molecular features resolved by MS-DIAL, among which >20,000 correlated with urban sources in Dhaka. A major wastewater treatment plant was not a dominant pollution source, consistent with major contributions from uncontrolled urban drainage, a result that encourages development of further wastewater infrastructures. Matching of deconvoluted MS2 spectra to public libraries resulted in 62 confident annotations (i.e., Level 1-2a) and allowed semiquantification of 42 analytes including pharmaceuticals, pesticides, and personal care products. In silico structure prediction for the top 100 unknown molecular features associated with an urban source allowed 15 additional chemicals of anthropogenic origin to be annotated (i.e., Level 3). The authentic MS2 spectra were uploaded to MassBank Europe, mass spectral data were openly shared on the MassIVE repository, a tool (i.e., MASST) that could be used for community science environmental surveillance was demonstrated, and current limitations were discussed.

Novel Per- and Polyfluoroalkyl Substances Discovered in Cattle Exposed to AFFF-Impacted Groundwater.

The leaching of per- and polyfluoroalkyl substances (PFASs) from Australian firefighting training grounds has resulted in extensive contamination of groundwater and nearby farmlands. Humans, farm animals, and wildlife in these areas may have been exposed to complex mixtures of PFASs from aqueous film-forming foams (AFFFs). This study aimed to identify PFAS classes in pooled whole blood (n = 4) and serum (n = 4) from cattle exposed to AFFF-impacted groundwater and potentially discover new PFASs in blood. Thirty PFASs were identified at various levels of confidence (levels 1a-5a), including three novel compounds: (i) perfluorohexanesulfonamido 2-hydroxypropanoic acid (FHxSA-HOPrA), (ii) methyl((perfluorohexyl)sulfonyl)sulfuramidous acid, and (iii) methyl((perfluorooctyl)sulfonyl)sulfuramidous acid, belonging to two different classes. Biotransformation intermediate, perfluorohexanesulfonamido propanoic acid (FHxSA-PrA), hitherto unreported in biological samples, was detected in both whole blood and serum. Furthermore, perfluoroalkyl sulfonamides, including perfluoropropane sulfonamide (FPrSA), perfluorobutane sulfonamide (FBSA), and perfluorohexane sulfonamide (FHxSA) were predominantly detected in whole blood, suggesting that these accumulate in the cell fraction of blood. The suspect screening revealed several fluoroalkyl chain-substituted PFAS. The results suggest that targeting only the major PFASs in the plasma or serum of AFFF-exposed mammals likely underestimates the toxicological risks associated with exposure. Future studies of AFFF-exposed populations should include whole-blood analysis with high-resolution mass spectrometry to understand the true extent of PFAS exposure.

Associations between the chemical exposome and pregnancy induced hypertension.

Exposure to environmental chemicals has been linked to an increased risk of pregnancy-induced hypertension (PIH). This prospective cohort study examined the associations between PIH and maternal chemical exposure to four classes of chemicals (i.e., phthalates, bisphenols, perfluoroalkyl acids, non-essential metals and trace minerals). Participants included 420 pregnant women from the Alberta Pregnancy Outcomes and Nutrition (APrON) cohort who had data available on diagnosed PIH and environmental chemical exposure. Twelve phthalate metabolites, two bisphenols, eight perfluoroalkyl acids and eleven non-essential metals or trace minerals were quantified in maternal urine or blood samples collected in the second trimester of pregnancy. Associations between the urinary and blood concentrations of these chemicals and PIH were assessed using multiple logistic and LASSO regression analyses in single- and multi-chemical exposure models, respectively. Thirty-five (8.3%) participants were diagnosed with PIH. In single chemical exposure models, two phthalate metabolites, mono-methyl phthalate (MMP) and monoethyl phthalate (MEP), three perfluoroalkyl acids, perfluoroheptanoic acid (PFHpA), perfluorononanoic acid (PFNA), and perfluorodecanoic acid (PFDA), and one metal, manganese, were associated with increased odds of PIH. The metabolites of di (2-ethylhexyl) phthalate (DEHP) and the molar sum of these metabolites, as well as antimony, displayed trend associations (p < 0.10). In multi-chemical exposure models using LASSO penalized regressions and double-LASSO regressions, MEP (AOR: 1.43, 95% CI: 1.09-1.88, p = 0.009) and PFNA (AOR: 2.03, 95% CI: 1.01-4.07, p = 0.04) were selected as the chemicals most highly associated with PIH. These findings suggest that maternal levels of phthalates and perfluoroalkyl acids may be associated with the diagnosis on PIH. Future research should consider both individual and multi-chemical exposures when examining predictors of PIH and other maternal cardiometabolic health disorders, such as preeclampsia, eclampsia, HELLP syndrome, and gestational diabetes.

Complex impacts of hydraulic fracturing return fluids on soil microbial community respiration, structure and functional potentials.

The consequences of soils exposed to hydraulic fracturing (HF) return fluid, often collectively termed flowback and produced water (FPW), are poorly understood, even though soils are a common receptor of FPW spills. Here, we investigate the impacts on soil microbiota exposed to FPW collected from the Montney Formation of western Canada. We measured soil respiration, microbial community structure and functional potentials under FPW exposure across a range of concentrations, exposure time and soil types (luvisol and chernozem). We find that soil type governs microbial community response upon FPW exposure. Within each soil, FPW exposure led to reduced biotic soil respiration, and shifted microbial community structure and functional potentials. We detect substantially higher species richness and more unique functional genes in FPW-exposed soils than in FPW-unexposed soils, with metagenome-assembled genomes (e.g. Marinobacter persicus) from luvisol soil exposed to concentrated FPW being most similar to genomes from HF/FPW sites. Our data demonstrate the complex impacts of microbial communities following FPW exposure and highlight the site-specific effects in evaluation of spills and agricultural reuse of FPW on the normal soil functions.

MS2Tox Machine Learning Tool for Predicting the Ecotoxicity of Unidentified Chemicals in Water by Nontarget LC-HRMS.

To achieve water quality objectives of the zero pollution action plan in Europe, rapid methods are needed to identify the presence of toxic substances in complex water samples. However, only a small fraction of chemicals detected with nontarget high-resolution mass spectrometry can be identified, and fewer have ecotoxicological data available. We hypothesized that ecotoxicological data could be predicted for unknown molecular features in data-rich high-resolution mass spectrometry (HRMS) spectra, thereby circumventing time-consuming steps of molecular identification and rapidly flagging molecules of potentially high toxicity in complex samples. Here, we present MS2Tox, a machine learning method, to predict the toxicity of unidentified chemicals based on high-resolution accurate mass tandem mass spectra (MS2). The MS2Tox model for fish toxicity was trained and tested on 647 lethal concentration (LC50) values from the CompTox database and validated for 219 chemicals and 420 MS2 spectra from MassBank. The root mean square error (RMSE) of MS2Tox predictions was below 0.89 log-mM, while the experimental repeatability of LC50 values in CompTox was 0.44 log-mM. MS2Tox allowed accurate prediction of fish LC50 values for 22 chemicals detected in water samples, and empirical evidence suggested the right directionality for another 68 chemicals. Moreover, by incorporating structural information, e.g., the presence of carbonyl-benzene, amide moieties, or hydroxyl groups, MS2Tox outperforms baseline models that use only the exact mass or log KOW.

Adult Exposure to Di-N-Butyl Phthalate (DBP) Induces Persistent Effects on Testicular Cell Markers and Testosterone Biosynthesis in Mice.

Studies indicate that phthalates are endocrine disruptors affecting reproductive health. One of the most commonly used phthalates, di-n-butyl phthalate (DBP), has been linked with adverse reproductive health outcomes in men, but the mechanisms behind these effects are still poorly understood. Here, adult male mice were orally exposed to DBP (10 or 100 mg/kg/day) for five weeks, and the testis and adrenal glands were collected one week after the last dose, to examine more persistent effects. Quantification of testosterone, androstenedione, progesterone and corticosterone concentrations by liquid chromatography-mass spectrometry showed that testicular testosterone was significantly decreased in both DBP treatment groups, whereas the other steroids were not significantly altered. Western blot analysis of testis revealed that DBP exposure increased the levels of the steroidogenic enzymes CYP11A1, HSD3ß2, and CYP17A1, the oxidative stress marker nitrotyrosine, and the luteinizing hormone receptor (LHR). The analysis further demonstrated increased levels of the germ cell marker DAZL, the Sertoli cell markers vimentin and SOX9, and the Leydig cell marker SULT1E1. Overall, the present work provides more mechanistic understanding of how adult DBP exposure can induce effects on the male reproductive system by affecting several key cells and proteins important for testosterone biosynthesis and spermatogenesis, and for the first time shows that these effects persist at least one week after the last dose. It also demonstrates impairment of testosterone biosynthesis at a lower dose than previously reported.

Postnatal BPA is associated with increasing executive function difficulties in preschool children.

BACKGROUND: Early bisphenol exposure may have consequences for executive function development, but less is known about potential sex effects. We hypothesized that early bisphenol A (BPA) and bisphenol S (BPS) exposures would be associated with sex-dependent changes in preschool executive function. METHODS: A subsample of the Alberta Pregnancy Outcomes and Nutrition (APrON) cohort (n = 312) provided maternal second trimester (prenatal) and 3-month postpartum (postnatal) urine samples, from which BPA and BPS concentrations were quantified. When children were age 2 and 4, mothers completed the Behavior Rating Inventory of Executive Function-Preschool Version (BRIEF-P). Changes in standardized T scores on the BRIEF-P indexes of inhibitory self-control, flexibility, and emergent metacognition were investigated. RESULTS: Adjusted multivariate regression analyses showed that child sex modified the associations between maternal postnatal BPA and changes in executive function. Higher maternal postnatal BPA concentrations predicted increasing difficulties from age 2 to 4 in the domains of inhibitory self-control and emergent metacognition in female, but not male children. The other bisphenol concentrations were not associated with changes in executive function. CONCLUSION: Due to the ubiquity of BPA exposure among breastfeeding women, these findings justify further investigation on the effects of postnatal bisphenol exposure on child cognitive development. IMPACT: Higher concentrations of maternal BPA at 3-month postpartum were associated with increasing difficulties in inhibitory self-control and emergent metacognition from age 2 to 4 in girls, but not boys. Prenatal BPA and prenatal/postnatal BPS were not significant predictors of changes in executive function in boys and girls. The current study extends previous research to show that maternal postnatal BPA could also impact child executive function. Due to the ubiquity of BPA exposure among breastfeeding women, the current findings suggest that additional precautions may be needed to protect infants' neurodevelopment from indirect exposure to BPA.

Non-target profiling of bitumen-influenced waters for the identification of tracers unique to oil sands processed-affected water (OSPW) in the Athabasca watershed of Alberta, Canada.

RATIONALE: The objective of this study was to identify unique chemical tracers of oil sands process-affected water (OSPW) to enable definitive discrimination of tailings pond seepage from natural bitumen-influenced waters from the Canadian Alberta McMurray formation. METHODS: The approach involved comparing unknowns from an unprecedented sample set of OSPW (n = 4) and OSPW-affected groundwaters (n = 15) with natural bitumen-influenced groundwaters (n = 20), using high-performance liquid chromatography/electrospray ionisation high-resolution mass spectrometry (HPLC/ESI-HRMS) operated in both polarities. RESULTS: Four unknown chemical entities were identified as potential tracers of OSPW seepage and subsequently subjected to structural elucidation. One potential tracer, tentatively identified as a thiophene-containing carboxylic acid [C15 H23 O3 S]- , was only detected in OSPW and OSPW-affected samples, thereby showing the greatest diagnostic potential. The remaining three unknowns, postulated to be two thiochroman isomers [C17 H25 O3 S]+ and an ethyl-naphthalene isomer [C16 H21 ]+ , were detected in one and two background groundwaters, respectively. CONCLUSIONS: We advanced the state of knowledge for tracers of tailings seepage beyond heteroatomic classes, to identifying diagnostic substances, with structures postulated. Synthesis of the four proposed structures is recommended to enable structural confirmations. This research will guide and inform the Oil Sands Monitoring Program in its efforts to assess potential influences of oil sands development on the Athabasca River watershed.

White matter microstructure mediates the association between prenatal exposure to phthalates and behavior problems in preschool children.

BACKGROUND: Previous research reports associations between prenatal exposure to phthalates and childhood behavior problems; however, the neural mechanisms that may underlie these associations are relatively unexplored. OBJECTIVE: This study examined microstructural white matter as a possible mediator of the associations between prenatal phthalate exposure and behavior problems in preschool-aged children. METHODS: Data are from a subsample of a prospective pregnancy cohort, the Alberta Pregnancy Outcomes and Nutrition (APrON) study (n = 76). Mother-child pairs were included if mothers provided a second trimester urine sample, if the child completed a successful magnetic resonance imaging (MRI) scan at age 3-5 years, and if the Child Behavior Checklist was completed within 6 months of the MRI scan. Molar sums of high (HMWP) and low molecular weight phthalates (LMWP) were calculated from levels in urine samples. Associations between prenatal phthalate concentrations, fractional anisotropy (FA) and mean diffusivity (MD) in 10 major white matter tracts, and preschool behavior problems were investigated. RESULTS: Maternal prenatal phthalate concentrations were associated with MD of the right inferior fronto-occipital fasciculus (IFO), right pyramidal fibers, left and right uncinate fasciculus (UF), and FA of the left inferior longitudinal fasciculus (ILF). Mediation analyses showed that prenatal exposure to HMWP was indirectly associated with Internalizing (path ab = 0.09, CI.95 = 0.02, 0.20) and Externalizing Problems (path ab = 0.09, CI.95 = 0.01, 0.19) through MD of the right IFO, and to Internalizing Problems (path ab = 0.11, CI.95 = 0.01, 0.23) through MD of the right pyramidal fibers. DISCUSSION: This study provides the first evidence of childhood neural correlates of prenatal phthalate exposure. Results suggest that prenatal phthalate exposure may be related to microstructural white matter in the IFO, pyramidal fibers, UF, and ILF. Further, MD of the right IFO and pyramidal fibers may transmit childhood risk for behavioral problems.

Neurodevelopmental and Metabolomic Responses from Prenatal Coexposure to Perfluorooctanesulfonate (PFOS) and Methylmercury (MeHg) in Sprague-Dawley Rats.

Methylmercury (MeHg) and perfluorooctanesulfonate (PFOS) are major contaminants of human blood that are both common in dietary fish, thereby raising questions about their combined impact on human development. Here, pregnant Sprague-Dawley rats ingested a daily dose, from gestational day 1 through to weaning, of either 1 mg/kg bw PFOS (PFOS-only), 1 mg/kg MeHg (MeHg-only), a mixture of 0.1 mg/kg PFOS and 1 mg/kg MeHg (Low-Mix), or of 1 mg/kg of PFOS and 1 mg/kg MeHg (High-Mix). Newborns were monitored for physical milestones and reflexive developmental responses, and in juveniles the spontaneous activity, anxiety, memory, and cognition were assessed. Targeted metabolomics of 199 analytes was applied to sectioned brain regions of juvenile offspring. Newborns in the High-Mix group had decreased weight gain as well as delayed reflexes and innate behavioral responses compared to controls and individual chemical groups indicating a toxicological interaction on early development. In juveniles, cumulative mixture effects increased in a dose-dependent manner in tests of anxiety-like behavior. However, other developmental test results suggested antagonism, as PFOS-only and MeHg-only juveniles had increased hyperactivity and thigmotaxic behavior, respectively, but fewer effects in Low-Mix and High-Mix groups. Consistent with these behavioral observations, a pattern of antagonism was also observed in neurochemicals measured in rat cortex, as PFOS-only and MeHg-only juveniles had altered concentrations of metabolites (e.g., lipids, amino acids, and biogenic amines), while no changes were evident in the combined exposures. The cortical metabolites altered in PFOS-only and MeHg-only exposed groups are involved in inhibitory and excitatory neurotransmission. These proof-of-principle findings at relatively high doses indicate the potential for toxicological interaction between PFOS and MeHg, with developmental-stage specific effects. Future mixture studies at lower doses are warranted, and prospective human birth cohorts should consider possible confounding effects from PFOS and mercury exposure on neurodevelopment.

Maternal exposure to arsenic and mercury in small-scale gold mining areas of Northern Tanzania.

Artisanal and small-scale gold mining (ASGM) in Tanzania results in occupational exposures and environmental contamination to toxic chemical elements such as arsenic and mercury. Populations living in such areas may be exposed by various routes, and prenatal exposure to arsenic and mercury has been associated with adverse birth outcomes and developmental delays. The aim of this study was to determine if levels of arsenic and mercury differed among pregnant women living in areas with and without ASGM activities in Northern Tanzania. This cross-sectional study is part of the ongoing Mining and Health prospective longitudinal study. Spot urine samples and dried blood spots were collected at the antenatal health clinics from pregnant women (n = 1056) at 16-27 weeks gestation. Urine samples were analyzed for total arsenic (T-As) and dried blood spots were analyzed for total mercury (T-Hg). Women in the ASGM cohort had median T-As levels (9.4 µg/L; IQR: 4.9-15.1) and T-Hg levels (1.2 µg/L; IQR: 0.8-1.86) that were significantly higher than the median T-As levels (6.28 µg/L; IQR: 3.7-14.1) and T-Hg levels (0.66 µg/L; IQR: 0.3-1.2) of women in the non-ASGM cohort (Mann-Whitney U test, T-As: z = -9.881, p = 0.0005; T-Hg: z = -3.502, p < 0.0001). Among pregnant women from ASGM areas, 25% had urinary T-As and 75% had blood T-Hg above the established human biomonitoring reference values of 15 and 0.80 µg/L. In the ASGM cohort, lower maternal education and low socioeconomic status increased the odds of higher T-As levels by 20% (p < 0.05) and 10% (p < 0.05), respectively. Women involved in mining activities and those of low socioeconomic status had increased odds of higher T-Hg by 70% (p < 0.001) and 10% (p < 0.05), respectively. Arsenic and mercury concentrations among women in non-ASGM areas suggest exposure sources beyond ASGM activities that need to be identified. Arsenic and mercury levels in women in Tanzania are of public health concern and their association with adverse birth and child developmental outcomes will be examined in future studies on this cohort.

Prenatal maternal and childhood bisphenol a exposure and brain structure and behavior of young children.

BACKGROUND: Bisphenol A (BPA) is commonly used in the manufacture of plastics and epoxy resins. In North America, over 90% of the population has detectable levels of urinary BPA. Human epidemiological studies have reported adverse behavioral outcomes with BPA exposure in children, however, corresponding effects on children's brain structure have not yet been investigated. The current study examined the association between prenatal maternal and childhood BPA exposure and white matter microstructure in children aged 2 to 5 years, and investigated whether brain structure mediated the association between BPA exposure and child behavior. METHODS: Participants were 98 mother-child pairs who were recruited between January 2009 and December 2012. Total BPA concentrations in spot urine samples obtained from mothers in the second trimester of pregnancy and from children at 3-4 years of age were analyzed. Children participated in a diffusion magnetic resonance imaging (MRI) scan at age 2-5 years (3.7 ± 0.8 years). Associations between prenatal maternal and childhood BPA and children's fractional anisotropy and mean diffusivity of 10 isolated white matter tracts were investigated, controlling for urinary creatinine, child sex, and age at the time of MRI. Post-hoc analyses examined if alterations in white matter mediated the relationship of BPA and children's scores on the Child Behavior Checklist (CBCL). RESULTS: Prenatal maternal urinary BPA was significantly associated with child mean diffusivity in the splenium and right inferior longitudinal fasciculus. Splenium diffusivity mediated the relationship between maternal prenatal BPA levels and children's internalizing behavior (indirect effect: ß = 0.213, CI [0.0167, 0.564]). No significant associations were found between childhood BPA and white matter microstructure. CONCLUSIONS: This study provides preliminary evidence for the neural correlates of BPA exposure in humans. Our findings suggest that prenatal maternal exposure to BPA may lead to alterations in white matter microstructure in preschool aged children, and that such alterations mediate the relationship between early life exposure to BPA and internalizing problems.

Toxicity in aquatic model species exposed to a temporal series of three different flowback and produced water samples collected from a horizontal hydraulically fractured well.

In the present study, we compared the toxicity and associated chemical characterizations of flowback and produced water (FPW) collected from a single horizontal hydraulically fractured well at different time points during FPW production. Since few studies on whole mixture toxicity related to FPW exist, our aims were to determine both overall toxicity of the FPW mixture in a suite of organisms (Daphnia magna, Lumbriculus variegatus, Danio rerio, and Oncorhynchus mykiss) and also determine if toxicity changes depending on variation in FPW chemical properties as a function of time sampled (1.33, 72, and 228 h FPW samples collected immediately post-well production onset were analyzed in current study). FPW chemical composition was determined via quadra-pole inductively coupled plasma - mass spectrometry/mass spectrometry (ICP-MS/MS), full-scan high performance liquid chromatography/Orbitrap mass spectrometry (HPLC/Orbitrap-MS), and gas chromatography-mass spectrometry (GC-MS). We observed that FPW sampled later in the production process contained higher ion and total dissolved solids concentrations, whereas the highest concentrations of dissolved organic compounds were observed in the earliest FPW sample analyzed. Toxicity associated with FPW exposure was deemed to be species-specific to a certain extent, but general trends revealed the earliest FPW sampled contained highest toxic potential. Accordingly, we theorize that although the saline conditions of FPW are the foremost toxicological drivers to freshwater organisms, dissolved organics associated with FPW significantly contribute to the overall toxicity of exposed organisms.