Concordance between In Vitro and In Vivo Relative Toxic Potencies of Diesel Exhaust Particles from Different Biodiesel Blends.

Diesel exhaust particles (DEPs) contribute to air pollution exposure-related adverse health impacts. Here, we examined in vitro, and in vivo toxicities of DEPs from a Caterpillar C11 heavy-duty diesel engine emissions using ultra-low-sulfur diesel (ULSD) and biodiesel blends (20% v/v) of canola (B20C), soy (B20S), or tallow-waste fry oil (B20T) in ULSD. The in vitro effects of DEPs (DEPULSD, DEPB20C, DEPB20S, and DEPB20T) in exposed mouse monocyte/macrophage cells (J774A.1) were examined by analyzing the cellular cytotoxicity endpoints (CTB, LDH, and ATP) and secreted proteins. The in vivo effects were assessed in BALB/c mice (n = 6/group) exposed to DEPs (250 µg), carbon black (CB), or saline via intratracheal instillation 24 h post-exposure. Bronchoalveolar lavage fluid (BALF) cell counts, cytokines, lung/heart mRNA, and plasma markers were examined. In vitro cytotoxic potencies (e.g., ATP) and secreted TNF-α were positively correlated (p < 0.05) with in vivo inflammatory potency (BALF cytokines, lung/heart mRNA, and plasma markers). Overall, DEPULSD and DEPB20C appeared to be more potent compared to DEPB20S and DEPB20T. These findings suggested that biodiesel blend-derived DEP potencies can be influenced by biodiesel sources, and inflammatory process- was one of the potential underlying toxicity mechanisms. These observations were consistent across in vitro and in vivo exposures, and this work adds value to the health risk analysis of cleaner fuel alternatives.

Prenatal exposure to perfluoroalkyl substances and inflammatory biomarker concentrations.

Per- and polyfluoroalkyl substances (PFAS) are persistent environmental contaminants that induce immunotoxicity in experimental studies; however, epidemiological evidence-particularly during pregnancy-is scarce. We quantified associations between first trimester plasma perfluorooctanoic acid (PFOA), perfluorooctane sulfonate (PFOS), and perfluorohexane sulfonate (PFHxS) concentrations and third trimester concentrations of inflammatory biomarkers and determined if these associations were modified by fetal sex. Methods: We analyzed data from 1411 participants, recruited between 2008 and 2011, in the Maternal-Infant Research on Environmental Chemicals study. Our primary outcome was a composite inflammatory index derived by summing the z-scores of eight proinflammatory biomarkers. Using multivariable linear regression models, we quantified associations between each PFAS and the inflammatory index and individual biomarkers. We quantified the effects of the PFAS mixture using weighted quantile sum regression, and evaluated effect modification using product terms and sex-stratified models. Results: Each doubling of PFOA and PFHxS was associated with a 0.38 (95% CI, 0.09, 0.67) and 0.21 (95% CI, 0.01, 0.41) SD increase in the proinflammatory index, respectively. A one-quartile increase in the PFAS mixture was associated with a 0.40 (95% CI, 0.09, 0.71) SD increase in the proinflammatory index. In individual models, we observed positive associations between PFAS and concentrations of monocyte chemoattractant protein-1, macrophage inflammatory protein-1ß, and matrix metalloproteinases-9; however, the magnitude and precision varied according to the specific PFAS. Sex-specific findings were identified in few PFAS-biomarker associations. Conclusions: PFOA, PFOS, and PFHxS, individually and as a mixture, were positively associated with proinflammatory biomarkers during pregnancy.

Association between toxic metals, vitamin D and preterm birth in the Maternal-Infant research on environmental chemicals study.

BACKGROUND: Toxic metals, like lead, are risk factors for preterm birth (PTB), but few studies have examined low levels found in most Canadians. Vitamin D, which may have antioxidant activity, protects against PTB. OBJECTIVES: In this study, we investigated the impact of toxic metals (lead, mercury, cadmium and arsenic) on PTB and examined if maternal plasma vitamin D concentrations modify these associations. METHODS: We investigated whether concentrations of metals in whole blood measured in early and late pregnancy were associated with PTB (<37 weeks) and spontaneous PTB in 1851 live births from the Maternal-Infant Research on Environmental Chemicals Study using discrete time survival analysis. We also investigated whether the risk of PTB was modified by first-trimester plasma 25-hydroxyvitamin D (25OHD) concentrations. RESULTS: Of 1851 live births, 6.1% (n = 113) were PTBs and 4.9% (n = 89) were spontaneous PTB. A 1 µg/dL increase in blood lead concentrations during pregnancy was associated with an increased risk of PTB (relative risk [RR] 1.48, 95% confidence interval [CI] 1.00, 2.20) and spontaneous PTB (RR 1.71, 95% CI 1.13, 2.60). The risk was higher in women with insufficient vitamin D concentrations (25OHD <50 nmol/L) for both PTB (RR 2.42, 95% CI 1.01, 5.79) and spontaneous PTB (RR 3.04, 95% CI 1.15, 8.04). However, an interaction on the additive scale was not present. Arsenic was associated with a higher risk of PTB (RR 1.10, 95% CI 1.02, 1.19) and spontaneous PTB (RR 1.11, 95% CI 1.03, 1.20) per 1 µg/L. CONCLUSIONS: Gestational exposure to low levels of lead and arsenic may increase the risk of PTB and spontaneous PTB; individuals with insufficient vitamin D may be more susceptible to the adverse effects of lead. Given our relatively small number of cases, we encourage testing of this hypothesis in other cohorts, especially those with vitamin D-deficient populations.

Adverse outcome pathways and linkages to transcriptomic effects relevant to ionizing radiation injury.

BACKGROUND: In the past three decades, a large body of data on the effects of exposure to ionizing radiation and the ensuing changes in gene expression has been generated. These data have allowed for an understanding of molecular-level events and shown a level of consistency in response despite the vast formats and experimental procedures being used across institutions. However, clarity on how this information may inform strategies for health risk assessment needs to be explored. An approach to bridge this gap is the adverse outcome pathway (AOP) framework. AOPs represent an illustrative framework characterizing a stressor associated with a sequential set of causally linked key events (KEs) at different levels of biological organization, beginning with a molecular initiating event (MIE) and culminating in an adverse outcome (AO). Here, we demonstrate the interpretation of transcriptomic datasets in the context of the AOP framework within the field of ionizing radiation by using a lung cancer AOP (AOP 272: https://www.aopwiki.org/aops/272) as a case example. METHODS: Through the mining of the literature, radiation exposure-related transcriptomic studies in line with AOP 272 related to lung cancer, DNA damage response, and repair were identified. The differentially expressed genes within relevant studies were collated and subjected to the pathway and network analysis using Reactome and GeneMANIA platforms. Identified pathways were filtered (p < .001, ≥3 genes) and categorized based on relevance to KEs in the AOP. Gene connectivities were identified and further grouped by gene expression-informed associated events (AEs). Relevant quantitative dose-response data were used to inform the directionality in the expression of the genes in the network across AEs. RESULTS: Reactome analyses identified 7 high-level biological processes with multiple pathways and associated genes that mapped to potential KEs in AOP 272. The gene connectivities were further represented as a network of AEs with associated expression profiles that highlighted patterns of gene expression levels. CONCLUSIONS: This study demonstrates the application of transcriptomics data in AOP development and provides information on potential data gaps. Although the approach is new and anticipated to evolve, it shows promise for improving the understanding of underlying mechanisms of disease progression with a long-term vision to be predictive of adverse outcomes.

In vitro toxicity screening of amorphous silica nanoparticles using mitochondrial fraction exposure followed by MS-based proteomic analysis.

Silica nanoparticles (SiNPs) are used in consumer products, engineering and medical technologies. Attractive properties of SiNPs (e.g. size/surface-modification) enhance usage and thus the likelihood of environmental/human exposures. The assessment of health risks associated with exposures to SiNPs requires information on their relative potencies and toxicity mechanisms. In this work, phagocytic J774 cells were exposed to amorphous pristine (15, 30, 75 nm) and surface-modified (-NH2, -C3COOH, -C11COOH, -PEG) SiNP variants, and internalization was assessed by transmission electron microscopy (TEM), while cellular ATP was measured as a cytotoxicity endpoint. Furthermore, mitochondrial fractions from J774 cells were exposed to these SiNP variants (5, 15 µg mL-1), as well as two reference particles (SiNP 12 nm and TiO2), and proteomic changes were analyzed by mass spectrometry. Ingenuity Pathway Analysis was used to identify toxicity pathways. TEM analyses showed SiNP internalization and distribution along with some changes in mitochondrial structure. SiNP size- and surface-modification and chemical composition-related changes in mitochondrial proteins, including key proteins of the respiratory complex and oxidative stress, were evident based on high content mass spectrometry data. In addition, the dose-related decrease in cellular ATP levels in SiNP-exposed cells was consistent with related mitochondrial protein profiles. These findings suggest that physicochemical properties can be determinants of SiNP exposure-related mitochondrial effects, and mitochondrial exposures combined with proteomic analysis can be valuable as a new approach methodology in the toxicity screening of SiNPs for risk assessment, with added insight into related toxicity mechanisms.

Blood metals and vitamin D status in a pregnancy cohort: A bidirectional biomarker analysis.

Low 25-hydroxyvitamin D (25OHD), a biomarker of vitamin D status, is associated with reduced immune function and adverse pregnancy outcomes, such as preterm birth. Observational studies indicate that long-term, high level exposure to metals such as cadmium (Cd) and lead (Pb) can impact a person's vitamin D status. However, the directionality of the association is uncertain, particularly for low-level exposures. We used three distinct longitudinal data analysis methods to investigate cross-sectional, longitudinal and bidirectional relationships of Cd and Pb biomarkers with 25-hydroxyvitamin D (25OHD) in a Canadian pregnancy cohort. Maternal whole blood Cd and Pb and plasma 25OHD concentrations were measured in the 1st (n = 1905) and 3rd (n = 1649) trimester and at delivery (25OHD only, n = 1542). Our multivariable linear regression analysis showed weak inverse associations between Cd and 25OHD concentrations cross-sectionally and longitudinally while the latent growth curve models showed weak associations with Pb on the 25OHD intercept. In the bidirectional analysis, using cross lagged panel models, we found no association between 1st trimester metals and 3rd trimester 25OHD. Instead, 1st trimester 25OHD was associated with 9% (-15%, -3%) lower 3rd trimester Cd and 3% (-7, 0.1%) lower Pb. These findings suggest the 25OHD may modify metal concentrations in pregnancy and demonstrates the value of controlling for contemporaneous effects and the persistence of a biomarker over time, in order to rule out reverse causation.

Acute cardiovascular effects of inhaled ambient particulate matter: Chemical composition-related oxidative stress, endothelin-1, blood pressure, and ST-segment changes in Wistar rats.

Short-term increases in particulate matter (PM) are associated with heightened morbidity and mortality from cardiovascular causes. Inhalation of PM is known to increase endothelin (ET)-1 levels. Yet, less is known about particle composition-related changes at the molecular level including the endothelinergic system and relationship with cardiovascular function changes. In this work, adult Wistar male rats were exposed for 4 h by nose-only inhalation to clean air, Ottawa urban particles (EHC-93, 48 mg/m3) and water-leached (EHC-93L, 49 mg/m3) particles, to examine the effect of particle compositional changes on oxidative stress, circulating ETs, blood pressure, and heart electrophysiology. Particle deposition in the respiratory compartment was estimated at 85 µg (25 ng/cm2). Lung cell proliferation was low in both treatment groups, indicating absence of acute injury. Inhalation of EHC-93 caused statistically significant elevations (p < 0.05) of oxidative stress markers, ET-1, ET-3, blood pressure, and a decrease of ST-segment duration in the ECG at 1.5 days post-exposure. Leached particles (EHC-93L) caused rapid but transient elevation (p < 0.05) of oxidative stress, ET-1, ET-2, and ET-3 at earlier time points, with no changes in blood pressure or ST-segment. These results demonstrate that inhalation of urban particles at an internal dose inadequate to cause acute lung injury can induce oxidative stress, enhance vasoactive endothelins, leading to vasopressor response, affecting cardiac electrophysiology in Wistar rats, consistent with the cardiovascular impacts of ambient particles in human populations. Change in particle potency after removal of soluble species, notably cadmium, zinc and polar organics suggests that the toxicodynamics of cardiovascular effects can be modified by physicochemical properties of particles.

Ambient air pollution and inflammatory effects in a Canadian pregnancy cohort.

BACKGROUND: Epidemiologic studies have consistently reported associations between air pollution and pregnancy outcomes including preeclampsia and gestational diabetes. However, the biologic mechanisms underlying these relationships remain unclear as few studies have collected relevant biomarker data. We examined relationships between ambient PM2.5 and NO2 with markers of inflammation during pregnancy in a prospective cohort of Canadian women. METHODS: We analyzed data from 1170 women enrolled in the Maternal-Infant Research on Environmental Chemicals study. Daily residential PM2.5 and NO2 exposures during pregnancy were estimated using satellite-based and land-use regression models and used to create 14-day and 30-day exposure windows before blood-draw. Inflammatory markers C-reactive protein, interleukin-6, interleukin-8, and tumor necrosis factor-α were measured in third trimester plasma samples. Multivariable linear regression was used to estimate associations for an interquartile range (IQR) increase in PM2.5 and NO2 and markers of inflammation, while adjusting for individual-level confounders. RESULTS: Fourteen-day (IQR: 6.85 µg/m3) and 30-day (IQR: 6.15 µg/m3) average PM2.5 exposures before blood-draw were positively associated with C-reactive protein after adjustment for covariates (24.6% [95% CI = 9.4, 41.9] and 17.4% [95% CI = 1.0, 35.0] increases, respectively). This association was found to be robust in several sensitivity analyses. Neither PM2.5 nor NO2 exposures were associated with interleukin-6, interleukin-8, or tumor necrosis factor-α. CONCLUSION: Exposure to ambient PM2.5 is positively associated with maternal inflammatory pathways in late pregnancy. This may contribute to positive associations between ambient PM2.5 and risk of adverse pregnancy outcomes.

Toxic Metal Species and 'Endogenous' Metalloproteins at the Blood-Organ Interface: Analytical and Bioinorganic Aspects.

Globally, human exposure to environmental pollutants causes an estimated 9 million deaths per year and it could also be implicated in the etiology of diseases that do not appear to have a genetic origin. Accordingly, there is a need to gain information about the biomolecular mechanisms that causally link exposure to inorganic environmental pollutants with distinct adverse health effects. Although the analysis of blood plasma and red blood cell (RBC) cytosol can provide important biochemical information about these mechanisms, the inherent complexity of these biological matrices can make this a difficult task. In this perspective, we will examine the use of metalloentities that are present in plasma and RBC cytosol as potential exposure biomarkers to assess human exposure to inorganic pollutants. Our primary objective is to explore the principal bioinorganic processes that contribute to increased or decreased metalloprotein concentrations in plasma and/or RBC cytosol. Furthermore, we will also identify metabolites which can form in the bloodstream and contain essential as well as toxic metals for use as exposure biomarkers. While the latter metal species represent useful biomarkers for short-term exposure, endogenous plasma metalloproteins represent indicators to assess the long-term exposure of an individual to inorganic pollutants. Based on these considerations, the quantification of metalloentities in blood plasma and/or RBC cytosol is identified as a feasible research avenue to better understand the adverse health effects that are associated with chronic exposure of various human populations to inorganic pollutants. Exposure to these pollutants will likely increase as a consequence of technological advances, including the fast-growing applications of metal-based engineering nanomaterials.

In vitro exposure of human lens epithelial cells to X-rays at varied dose-rates leads to protein-level changes relevant to cataractogenesis.

BACKGROUND: Accumulated body of evidence shows that ionizing radiation increases the risk of cataracts. The mechanisms are not clear and the International Commission on Radiological Protection indicates a need for research into understanding the process, particularly at low doses and low dose rates of exposure. PURPOSE: This study was designed to examine protein-level modifications in a human lens epithelial (HLE) cell-line following radiation exposures. MATERIALS AND METHODS: HLE cell-line was subjected to X-irradiation at varied doses (0-5 Gy) and dose-rates (1.62 cGy/min and 38.2 cGy/min). Cells were collected 20 h post-exposure, lysed and proteins were clarified following fractionation by a molecular weight cut-off filtration method. Fractionated cellular proteins were enzymatically digested and subjected to mass spectrometry analysis. RESULTS: Statistically significant radiation dose-related protein changes compared to the control group were identified. Heatmap and hierarchical clustering analysis showed dose-rate dependant responses. Pathway analysis mapped the proteins to biological functions of mitochondrial dysfunction, reactive oxygen species generation, cell death, cancer, organismal injury and amyloidosis. CONCLUSION: Overall findings suggest that ionizing radiation exposure of HLE cells by mediating dose rate-dependant oxidative stress and cell death-related mechanisms, can be relevant to cataractogenesis.

Effect of industrial point-source air pollutants on fractional exhaled nitric oxide in healthy volunteers.

BACKGROUND: Few studies have examined the effects of industrial, fixed-site sources of air pollution on lung inflammation in nearby residents. We investigated the effects of short-term exposure to ambient air near a steel plant on the fractional exhaled concentration of nitric oxide (FeNO), a measure of airway inflammation, in healthy volunteers. METHODS: A cross-over study design was used. Fifty-nine non-smoking participants (mean age 24 years) were randomly assigned to each of two 5-day exposure scenarios: breathing ambient air adjacent to a steel plant or 5 km away at a college campus site. FeNO and on-site air pollutants were measured daily. Mixed effects linear regression models were used for data analysis, adjusting for sex, temperature, humidity and day of week. RESULTS: Compared with the college site, PM 2.5, ultrafine PM, SO2, NO2 and CO levels were significantly greater near the steel plant. FeNO was 15.3% (95% CI, 6.6%, 24.8%) higher near the plant compared to the college site. CONCLUSIONS: Exposure to ambient air near a steel plant was associated with increased airway inflammation as measured by exhaled nitric oxide.

Oxidative and Toxicological Evolution of Engineered Nanoparticles with Atmospherically Relevant Coatings.

The health impacts associated with engineered nanoparticles (ENPs) released into the atmosphere have not been adequately assessed. Such impacts could potentially arise from the toxicity associated with condensable atmospheric secondary organic material (SOM), or changes in the SOM composition induced by ENPs. Here, these possibilities are evaluated by investigating the oxidative and toxicological evolution of TiO2 and SiO2 nanoparticles which have been coated with SOM from the O3 or OH initiated oxidation of α-pinene. It was found that pristine SiO2 particles were significantly more cytotoxic compared to pristine TiO2 particles. TiO2 in the dark or under UV irradiation catalytically reacted with the SOM, increasing its O/C by up to 55% over photochemically inert SiO2 while having negligible effects on the overall cytotoxicity. Conversely, the cytotoxicity associated with SiO2 coated with SOM was markedly suppressed (by a factor of 9, at the highest exposure dose) with both increased SOM coating thickness and increased photochemical aging. These suppressing effects (organic coating and photo-oxidation of organics) were attributed to a physical hindrance of SiO2-cell interactions by the SOM and enhanced SOM viscosity and hydrophilicity with continued photo-oxidation, respectively. These findings highlight the importance of atmospheric processes in altering the cytotoxicity of ENPs.

Characterization of maternal plasma biomarkers associated with delivery of small and large for gestational age infants in the MIREC study cohort.

OBJECTIVE: Neonatal morbidity and mortality can be influenced by maternal health status. Information on maternal and fetal biomarkers of adverse health outcomes is limited. This work aims at identifying maternal biomarkers associated with low and high birth weight for gestational age groups. DESIGN AND SETTINGS: Population-based prospective cohort study of the potential adverse health effects of exposure to environmental contaminants on pregnancy and infant health. METHODS: Third trimester maternal plasma samples (n = 1588) from a pregnancy cohort (Maternal-Infant Research on Environmental Chemicals Study, MIREC) were analyzed for changes in a target spectrum of biomarkers of vascular health (e.g., matrix metalloproteinases MMPs, vascular endothelial cell growth factor VEGF), inflammation (e.g. cellular adhesion molecules CAMs, cytokines, chemokines) by affinity-based multiplex protein array analyses. Multivariate logistic regression analyses were done to examine associations between target plasma biomarkers, maternal-infant characteristics, and birth weight outcomes assessed as small for gestational age (SGA) ≤10th percentile and large for gestational age (LGA) ≥90th percentile groups. RESULTS AND OUTCOMES: Our results revealed that maternal plasma biomarkers monocyte chemoattractant protein-1 MCP-1 (p<0.05, +ve) and VEGF (p<0.05, -ve) along with parity = 1 (p<0.01, -ve) and gestational hypertension (p<0.05, +ve) were associated with SGA births. Meanwhile, LGA was associated with maternal plasma VEGF (p<0.05, +ve) and MMP-9 (p<0.05, -ve) and gestational hypertension (p<0.01, +ve), pre-pregnancy body mass index (p<0.01, +ve), parity (p<0.05, +ve) and education (p<0.05, -ve). CONCLUSIONS: Third trimester maternal plasma biomarkers in combination with maternal health and socioeconomic characteristics can be useful in predicting SGA and LGA outcomes. Maternal vascular health and inflammatory status may contribute to both SGA and LGA births through distinct molecular mechanisms.

Cardiovascular and inflammatory mechanisms in healthy humans exposed to air pollution in the vicinity of a steel mill.

BACKGROUND: There is a paucity of mechanistic information that is central to the understanding of the adverse health effects of source emission exposures. To identify source emission-related effects, blood and saliva samples from healthy volunteers who spent five days near a steel plant (Bayview site, with and without a mask that filtered many criteria pollutants) and at a well-removed College site were tested for oxidative stress, inflammation and endothelial dysfunction markers. METHODS: Biomarker analyses were done using multiplexed protein-array, HPLC-Fluorescence, EIA and ELISA methods. Mixed effects models were used to test for associations between exposure, biological markers and physiological outcomes. Heat map with hierarchical clustering and Ingenuity Pathway Analysis (IPA) were used for mechanistic analyses. RESULTS: Mean CO, SO2 and ultrafine particles (UFP) levels on the day of biological sampling were higher at the Bayview site compared to College site. Bayview site exposures "without" mask were associated with increased (p < 0.05) pro-inflammatory cytokines (e.g IL-4, IL-6) and endothelins (ETs) compared to College site. Plasma IL-1ß, IL-2 were increased (p < 0.05) after Bayview site "without" compared to "with" mask exposures. Interquartile range (IQR) increases in CO, UFP and SO2 were associated with increased (p < 0.05) plasma pro-inflammatory cytokines (e.g. IL-6, IL-8) and ET-1(1-21) levels. Plasma/saliva BET-1 levels were positively associated (p < 0.05) with increased systolic BP. C-reactive protein (CRP) was positively associated (p < 0.05) with increased heart rate. Protein network analyses exhibited activation of distinct inflammatory mechanisms after "with" and "without" mask exposures at the Bayview site relative to College site exposures. CONCLUSIONS: These findings suggest that air pollutants in the proximity of steel mill site can influence inflammatory and vascular mechanisms. Use of mask and multiple biomarker data can be valuable in gaining insight into source emission-related health impacts.

Oxidative and nitrative stress-related changes in human lens epithelial cells following exposure to X-rays.

PURPOSE: There is limited understanding of the mechanistic effects of ionizing radiation (IR) exposure in cataract formation. In this study, we explored the effects of IR on reactive oxygen/nitrogen species (ROS and RNS) generation in human lens epithelial (HLE) cells as an early key event to long-term damage. MATERIALS AND METHODS: HLE cell-line was exposed to X-rays at varied doses (0-5 Gy) and dose-rates. Cell lysates and supernatants were collected 20 h post-exposure and analysed for viability, cell cycling and metabolites of ROS (p, m-, o-, tyrosines, 3-chlorotyrosine (cl-tyrosine), 8-hydroxy deoxyguanosine, (8-OH-dG) and RNS (3-nitrotyrosine). RESULTS AND CONCLUSIONS: HLE cell-line exhibited a bi-phasic response in terms of cell viability, ROS and RNS profiles. At doses <0.5 Gy, ROS and RNS levels were lower than control and at higher doses (>0.5 Gy) a steady increase was observed in each metabolite. This response was observed irrespective of dose-rate. Among the associations tested, cl, p, m-tyrosine and 3-nitrotyrosine revealed changes (p < .05) at 5 Gy compared exclusively to 0.05 and 0.01 Gy. In addition, dose-rate related differences were observed. Overall, the data suggests that ROS and RNS are key events in radiation induced damage and this response is dependent on the dose and dose-rate of IR exposure.

In vitro toxicoproteomic analysis of A549 human lung epithelial cells exposed to urban air particulate matter and its water-soluble and insoluble fractions.

BACKGROUND: Toxicity of airborne particulate matter (PM) is difficult to assess because PM composition is complex and variable due to source contribution and atmospheric transformation. In this study, we used an in vitro toxicoproteomic approach to identify the toxicity mechanisms associated with different subfractions of Ottawa urban dust (EHC-93). METHODS: A549 human lung epithelial cells were exposed to 0, 60, 140 and 200 µg/cm2 doses of EHC-93 (total), its insoluble and soluble fractions for 24 h. Multiple cytotoxicity assays and proteomic analyses were used to assess particle toxicity in the exposed cells. RESULTS: The cytotoxicity data based on cellular ATP, BrdU incorporation and LDH leakage indicated that the insoluble, but not the soluble, fraction is responsible for the toxicity of EHC-93 in A549 cells. Two-dimensional gel electrophoresis results revealed that the expressions of 206 protein spots were significantly altered after particle exposures, where 154 were identified by MALDI-TOF-TOF-MS/MS. The results from cytotoxicity assays and proteomic analyses converged to a similar finding that the effects of the total and insoluble fraction may be alike, but their effects were distinguishable, and their effects were significantly different from the soluble fraction. Furthermore, the toxic potency of EHC-93 total is not equal to the sum of its insoluble and soluble fractions, implying inter-component interactions between insoluble and soluble materials resulting in synergistic or antagonistic cytotoxic effects. Pathway analysis based on the low toxicity dose (60 µg/cm2) indicated that the two subfractions can alter the expression of those proteins involved in pathways including cell death, cell proliferation and inflammatory response in a distinguishable manner. For example, the insoluble and soluble fractions differentially affected the secretion of pro-inflammatory cytokines such as MCP-1 and IL-8 and distinctly altered the expression of those proteins (e.g., TREM1, PDIA3 and ENO1) involved in an inflammatory response pathway in A549 cells. CONCLUSIONS: This study demonstrated the impact of different fractions of urban air particles constituted of various chemical species on different mechanistic pathways and thus on cytotoxicity effects. In vitro toxicoproteomics can be a valuable tool in mapping these differences in air pollutant exposure-related toxicity mechanisms.

Exposure to air pollution near a steel plant is associated with reduced heart rate variability: a randomised crossover study.

BACKGROUND: Epidemiological studies have shown that as ambient air pollution (AP) increases the risk of cardiovascular mortality also increases. The mechanisms of this effect may be linked to alterations in autonomic nervous system function. We wished to examine the effects of industrial AP on heart rate variability (HRV), a measure of subtle changes in heart rate and rhythm representing autonomic input to the heart. METHODS: Sixty healthy adults were randomized to spend five consecutive 8-h days outdoors in one of two locations: (1) adjacent to a steel plant in the Bayview neighbourhood in Sault Ste Marie Ontario or (2) at a College campus, several kilometers from the plant. Following a 9-16 day washout period, participants spent five consecutive days at the other site. Ambient AP levels and ambulatory electrocardiogram recordings were collected daily. HRV analysis was undertaken on a segment of the ambulatory ECG recording during a 15 min rest period, near the end of the 8-h on-site day. Standard HRV parameters from both time and frequency domains were measured. Ambient AP was measured with fixed site monitors at both sites. Statistical analysis was completed using mixed-effects models. RESULTS: Compared to the College site, HRV was statistically significantly reduced at the Bayview site by 13% (95%CI 3.6,19.2) for the standard deviation of normal to normal, 8% (95%CI 0.1, 4.9) for the percent normal to normal intervals differing by more than 50 ms, and 15% (95%CI 74.9, 571.2) for low frequency power. Levels of carbon monoxide, sulphur dioxide, nitrogen dioxide, and fine and ultrafine particulates were slightly, but statistically significantly, elevated at Bayview when compared to College. Interquartile range changes in individual air pollutants were significantly associated with reductions in HRV measured on the same day. The patterns of effect showed a high degree of consistency, with nearly all pollutants significantly inversely associated with at least one measure of HRV. CONCLUSIONS: The significant associations between AP and changes in HRV suggest that ambient AP near a steel plant may impact autonomic nervous system control of the heart.

Ser422 phosphorylation blocks human Tau cleavage by caspase-3: Biochemical implications to Alzheimer's Disease.

Proteolytic truncation of microtubule associated human (h) Tau protein by caspase-3 at the carboxy (C) terminus has been linked to the pathogenesis of Alzheimer's Disease (AD). This cleavage likely occurs between Asp421↓Ser422 leading to the formation of 421-mer truncated Tau protein which has been found to be present as aggregate in high level after phosphorylation in mortal AD brain tissue compared to normal. At least 50 phosphorylation sites involving Ser, Thr and Tyr residues have been identified or proposed in hTau and a selected number of them have been implicated in hTau aggregation following latter's proteolytic truncation. Interestingly, it is further noted that Ser422 residue present in the P1' position of hTau caspase-3 cleavage region is a potential phosphorylation site. So we became interested to examine in vitro the effect of phospho-Ser422 residue on hTau cleavage by caspase-3 which is a crucial upstream event associated with hTau self-assembly leading to AD pathogenesis. The goal of this project is to study in vitro the caspase-3 cleavage site of hTau protein and to examine the kinetics of this cleavage following Ser422 phosphorylation and treatment with caspase-3 inhibitors. This is achieved by designing peptides from the sequence of hTau protein containing the proposed caspase-3 cleavage region. Peptides were designed from 441-mer major human Tau protein sequence that encompasses the proposed caspase-3 cleavage site [Asp421↓Ser422]. Corresponding phospho-, dextro-Ser422 and dextro-Asp421 analogs were also designed. Peptides were synthesized by solid phase chemistry, purified and fully characterized by mass spectrometry. These were then incubated with recombinant caspase-3 enzyme under identical condition for digestion and analyzed for cleavage by mass spectrometry and RP-HPLC chromatograms. Our results indicated that while the control peptide is efficiently cleaved by caspase-3 at Asp421↓Ser422 site producing the expected N- and C-terminal fragment peptides, the corresponding phospho-Ser422 peptide remained completely resistant to the cleavage. Substitution of Asp421 by its dextro isoform also blocks peptide cleavage by caspase-3. However substitution of Ser422 by its dextro isoform in the peptide did not affect the cleavage significantly. The above results were further confirmed by caspase-3 digestion experiment in the presence of varying amounts of caspase-3 inhibitor (Ac-DQVD-aldehyde) which was found to block this cleavage in a highly effective manner. Our results highlighted the crucial significance of Ser422 phosphorylation and suggest that the kinase associated with this Ser-phosphorylation may protect Tau from aggregation. Thus specific promoters/activators of this kinase may find useful therapeutic benefits in arresting Tau truncation by caspase-3 and the progression of AD. In addition our data demonstrated that Tau-peptides where Ser422 or Asp421 are substituted by their respective dextro isomers, exhibit different cleavage kinetics by caspase-3 and this may have important implications in therapeutic intervention of Tau aggregation and associated AD.

Responses of A549 human lung epithelial cells to cristobalite and α-quartz exposures assessed by toxicoproteomics and gene expression analysis.

In this study, we used cytotoxicity assays, proteomic and gene expression analyses to examine the difference in response of A549 cells to two silica particles that differ in physical properties, namely cristobalite (CR) and α-quartz (Min-U-Sil 5, MI). Cytotoxicity assays such as lactate dehydrogenase release, 5-bromo-2'-deoxyuridine incorporation and cellular ATP showed that both silica particles could cause cell death, decreased cell proliferation and metabolism in the A549 human lung epithelial cells. While cytotoxicity assays revealed little difference between CR and MI exposures, proteomic and gene expression analyses unveiled both similar and unique molecular changes in A549 cells. For instance, two-dimensional gel electrophoresis data indicated that the expression of proteins in the cell death (e.g., ALDH1A1, HTRA2 and PRDX6) and cell proliferation (e.g., FSCN1, HNRNPAB and PGK1) pathways were significantly different between the two silica particles. Reverse transcription-polymerase chain reaction data provided additional evidence supporting the proteomic findings. Preliminary assessment of the physical differences between CR and MI suggested that the extent of surface interaction between particles and cells could explain some of the observed biological effects. However, the differential dose-response curves for some other genes and proteins suggest that other physical attributes of particulate matter can also contribute to particulate matter-related cellular toxicity. Our results demonstrated that toxicoproteomic and gene expression analyses are sensitive in distinguishing subtle toxicity differences associated with silica particles of varying physical properties compared to traditional cytotoxicity endpoints. Copyright © 2016 Her Majesty the Queen in Right of Canada. Journal of Applied Toxicology published by John Wiley & Sons, Ltd.

Associations between urinary biomarkers of oxidative stress and air pollutants observed in a randomized crossover exposure to steel mill emissions.

The effects of industrial air pollution on human health have not been as thoroughly investigated as those of urban air pollution which originates mostly from automotive transport. To better assess the health impacts of point sources of industrial air pollution, a randomized crossover exposure study was conducted. Sixty one young and healthy volunteers were randomly assigned to spend five consecutive eight-hour days near a steel mill or at a location five kilometres away. After a nine or sixteen-day washout period, volunteers spent another five consecutive days at the second site. Meteorological conditions and air pollutants were monitored at both exposure sites. On each exposure day, the first morning urine was collected along with a second urine sample obtained immediately before leaving the exposure site at the end of the day. Urinary levels of biomarkers of oxidative stress 8-hydroxy-2'-deoxyguanosine (8-OHdG, a biomarker of oxidative DNA damage), malondialdehyde (MDA, a biomarker of lipid peroxidation), 8-isoprostane (8-IsoP, a bioactive metabolite resulting from the peroxidation of arachidonic acid) and Vascular Endothelial Growth Factor (VEGF, involved in response to oxidative stress) were measured. According to mixed-effects linear regression models, intra-individual variations in 8-OHdG urinary levels were significantly associated with exposure site, but surprisingly, lower levels were observed at the steel mill site. Delayed, temporally-defined associations with specific air pollutants were observed for 8-OHdG, 8-IsoP and VEGF. However, these associations were subtle, presented complex patterns and their biological consequences remain unclear.