Enriched housing differentially alters allostatic load and cardiopulmonary responses to wildfire-related smoke in male and female mice.

Living conditions are an important modifier of individual health outcomes and may lead to higher allostatic load (AL). However, housing-induced cardiovascular and immune effects contributing to altered environmental responsiveness remain understudied. This investigation was conducted to examine the influence of enriched (EH) versus depleted housing (DH) conditions on cardiopulmonary functions, systemic immune responses, and allostatic load in response to a single wildfire smoke (WS) exposure in mice. Male and female C57BL/6J mice were divided into EH or DH for 22 weeks, and cardiopulmonary assessments measured before and after exposures to either one-hr filtered air (FA) or flaming eucalyptus WS exposure. Male and female DH mice exhibited increased heart rate (HR) and left ventricular mass (LVM), as well as reduced stroke volume and end diastolic volume (EDV) one week following exposure to WS. Female DH mice displayed significantly elevated levels of IL-2, IL-17, corticosterone and hemoglobin A1c (HbA1c) following WS, while female in EH mice higher epinephrine levels were detected. Female mice exhibited higher AL than males with DH, which was potentiated post-WS exposure. Thus, DH increased susceptibility to extreme air pollution in a gender-dependent manner suggesting that living conditions need to be evaluated as a modifier of toxicological responses.

The effect of enriched versus depleted housing on eucalyptus smoke-induced cardiovascular dysfunction in mice.

Objectives: Living conditions play a major role in health and well-being, particularly for the cardiovascular and pulmonary systems. Depleted housing contributes to impairment and development of disease, but how it impacts body resiliency during exposure to environmental stressors is unknown. This study examined the effect of depleted (DH) versus enriched housing (EH) on cardiopulmonary function and subsequent responses to wildfire smoke. Materials and Methods: Two cohorts of healthy female mice, one of them surgically implanted with radiotelemeters for the measurement of electrocardiogram, body temperature (Tco) and activity, were housed in either DH or EH for 7 weeks. Telemetered mice were exposed for 1 h to filtered air (FA) and then flaming eucalyptus wildfire smoke (WS) while untelemetered mice, which were used for ventilatory assessment and tissue collection, were exposed to either FA or WS. Animals were continuously monitored for 5-7 days after exposure. Results: EH prevented a decrease in Tco after radiotelemetry surgery. EH mice also had significantly higher activity levels and lower heart rate during and after FA and WS. Moreover, EH caused a decreased number of cardiac arrhythmias during WS. WS caused ventilatory depression in DH mice but not EH mice. Housing enrichment also upregulated the expression of cardioprotective genes in the heart. Conclusions: The results of this study indicate that housing conditions impact overall health and cardiopulmonary function. More importantly, depleted housing appears to worsen the response to air pollution. Thus, non-chemical factors should be considered when assessing the susceptibility of populations, especially when it comes to extreme environmental events.

Burn pit-related smoke causes developmental and behavioral toxicity in zebrafish: Influence of material type and emissions chemistry.

Combustion of mixed materials during open air burning of refuse or structural fires in the wildland urban interface produces emissions that worsen air quality, contaminate rivers and streams, and cause poor health outcomes including developmental effects. The zebrafish, a freshwater fish, is a useful model for quickly screening the toxicological and developmental effects of agents in such species and elicits biological responses that are often analogous and predictive of responses in mammals. The purpose of this study was to compare the developmental toxicity of smoke derived from the burning of 5 different burn pit-related material types (plywood, cardboard, plastic, a mixture of the three, and the mixture plus diesel fuel as an accelerant) in zebrafish larvae. Larvae were exposed to organic extracts of increasing concentrations of each smoke 6-to-8-hr post fertilization and assessed for morphological and behavioral toxicity at 5 days post fertilization. To examine chemical and biological determinants of toxicity, responses were related to emissions concentrations of polycyclic hydrocarbons (PAH). Emissions from plastic and the mixture containing plastic caused the most pronounced developmental effects, including mortality, impaired swim bladder inflation, pericardial edema, spinal curvature, tail kinks, and/or craniofacial deformities, although all extracts caused concentration-dependent effects. Plywood, by contrast, altered locomotor responsiveness to light changes to the greatest extent. Some morphological and behavioral responses correlated strongly with smoke extract levels of PAHs including 9-fluorenone. Overall, the findings suggest that material type and emissions chemistry impact the severity of zebrafish developmental toxicity responses to burn pit-related smoke.

Depleted housing elicits cardiopulmonary dysfunction after a single flaming eucalyptus wildfire smoke exposure in a sex-specific manner in ApoE knockout mice.

Although it is well established that wildfire smoke exposure can increase cardiovascular morbidity and mortality, the combined effects of non-chemical stressors and wildfire smoke remains understudied. Housing is a non-chemical stressor that is a major determinant of cardiovascular health, however, disparities in neighborhood and social status have exacerbated the cardiovascular health gaps within the United States. Further, pre-existing cardiovascular morbidities, such as atherosclerosis, can worsen the response to wildfire smoke exposures. This represents a potentially hazardous interaction between inadequate housing and stress, cardiovascular morbidities, and worsened responses to wildfire smoke exposures. The purpose of this study was to examine the effects of enriched (EH) versus depleted (DH) housing on pulmonary and cardiovascular responses to a single flaming eucalyptus wildfire smoke (WS) exposure in male and female apolipoprotein E (ApoE) knockout mice, which develop an atherosclerosis-like phenotype. The results of this study show that cardiopulmonary responses to WS exposure occur in a sex-specific manner. EH blunts adverse WS-induced ventilatory responses, specifically an increase in tidal volume (TV), expiratory time (Te), and relaxation time (RT) after a WS exposure, but only in females. EH also blunted a WS-induced increase in isovolumic relaxation time (IVRT) and the myocardial performance index (MPI) 1-wk after exposures, also only in females. Our results suggest that housing alters the cardiovascular response to a single WS exposure, and that DH might cause increased susceptibility to environmental exposures that manifest in altered ventilation patterns and diastolic dysfunction in a sex-specific manner.

Dose additive maternal and offspring effects of oral maternal exposure to a mixture of three PFAS (HFPO-DA, NBP2, PFOS) during pregnancy in the Sprague-Dawley rat.

Simultaneous exposure to multiple per- and polyfluoroalkyl substances (PFAS) is common in humans across the globe. Individual PFAS are associated with adverse health effects, yet the nature of mixture effects after exposure to two or more PFAS remains unclear. Previously we reported that oral administration of hexafluoropropylene oxide-dimer acid (HFPO-DA, or GenX), Nafion byproduct 2 (NBP2), or perfluorooctane sulfonate (PFOS) individually during pregnancy produced maternal and F1 effects. Here, we hypothesized that responses to the combined exposure to these three PFAS would be dose additive. Pregnant Sprague-Dawley rats were exposed to a fixed-ratio equipotent mixture where the top dose contained each PFAS at their ED50 for neonatal mortality (100 % dose = PFOS 3 mg/kg; NBP2 10 mg/kg; HFPO-DA 110 mg/kg), followed by a dilution series (33.3, 10, 3.3, and 1 %) and vehicle controls (0 % dose). Consistent with the single chemical studies, dams were exposed from gestation day (GD)14-18 or from GD8-postnatal day (PND2). Fetal and maternal livers on GD18 displayed multiple significantly upregulated genes associated with lipid and carbohydrate metabolism at all dose levels, while dams displayed significantly increased liver weight (≥3.3 % dose) and reduced serum thyroid hormones (≥33.3 % dose). Maternal exposure from GD8-PND2 significantly reduced pup bodyweights at birth (≥33.3 % dose) and PND2 (all doses), increased neonatal liver weights (≥3.3 % dose), increased pup mortality (≥3.3 % dose), and reduced maternal bodyweights and weight gain at the top dose. Echocardiography of adult F1 males and females identified significantly increased left ventricular anterior wall thickness (~10 % increase), whereas other cardiac morphological, functional, and transcriptomic measures were unaffected. Mixture effects in maternal and neonatal animals conformed to dose addition using a relative potency factor (RPF) analysis. Results support dose addition-based cumulative assessment approaches for estimating combined effects of PFAS co-exposure.

Mild allergic airways responses to an environmental mixture increase cardiovascular risk in rats.

Recent epidemiological findings link asthma to adverse cardiovascular responses. Yet, the precise cardiovascular impacts of asthma have been challenging to disentangle from the potential cardiovascular effects caused by asthma medication. The purpose of this study was to determine the impacts of allergic airways disease alone on cardiovascular function in an experimental model. Female Wistar rats were intranasally sensitized and then challenged once per week for 5 weeks with saline vehicle or a mixture of environmental allergens (ragweed, house dust mite, and Aspergillus fumigatus). Ventilatory and cardiovascular function, measured using double-chamber plethysmography and implantable blood pressure (BP) telemetry and cardiovascular ultrasound, respectively, were assessed before sensitization and after single and final allergen challenge. Responses to a single 0.5 ppm ozone exposure and to the cardiac arrhythmogenic agent aconitine were also assessed after final challenge. A single allergen challenge in sensitized rats increased tidal volume and specific airways resistance in response to provocation with methacholine and increased bronchoalveolar lavage fluid (BALF) eosinophils, neutrophils, lymphocytes, cytokines interleukin (IL)-4, IL-5, IL-10, IL-1ß, tumor necrosis factor-α, and keratinocyte chemoattract-growth-related oncogene characteristic of allergic airways responses. Lung responses after final allergen challenge in sensitized rats were diminished, although ozone exposure increased BALF IL-6, IL-13, IL-1 ß, and interferon-γ and modified ventilatory responses only in the allergen group. Final allergen challenge also increased systolic and mean arterial BP, stroke volume, cardiac output, end-diastolic volume, sensitivity to aconitine-induced cardiac arrhythmia, and cardiac gene expression with lesser effects after a single challenge. These findings demonstrate that allergic airways responses may increase cardiovascular risk in part by altering BP and myocardial function and by causing cardiac electrical instability.

Stress Drivers of Glucose Dynamics during Ozone Exposure Measured Using Radiotelemetry in Rats.

BACKGROUND: Inhaled irritant air pollutants may trigger stress-related metabolic dysfunction associated with altered circulating adrenal-derived hormones. OBJECTIVES: We used implantable telemetry in rats to assess real-time changes in circulating glucose during and after exposure to ozone and mechanistically linked responses to neuroendocrine stress hormones. METHODS: First, using a cross-over design, we monitored glucose during ozone exposures (0.0, 0.2, 0.4, and 0.8 ppm) and nonexposure periods in male Wistar Kyoto rats implanted with glucose telemeters. A second cohort of unimplanted rats was exposed to ozone (0.0, 0.4 or 0.8 ppm) for 30 min, 1 h, 2 h, or 4 h with hormones measured immediately post exposure. We assessed glucose metabolism in sham and adrenalectomized rats, with or without supplementation of adrenergic/glucocorticoid receptor agonists, and in a separate cohort, antagonists. RESULTS: Ozone (0.8 ppm) was associated with significantly higher blood glucose and lower core body temperature beginning 90 min into exposure, with reversal of effects 4-6 h post exposure. Glucose monitoring during four daily 4-h ozone exposures revealed duration of glucose increases, adaptation, and diurnal variations. Ozone-induced glucose changes were preceded by higher levels of adrenocorticotropic hormone, corticosterone, and epinephrine but lower levels of thyroid-stimulating hormone, prolactin, and luteinizing hormones. Higher glucose and glucose intolerance were inhibited in rats that were adrenalectomized or treated with adrenergic plus glucocorticoid receptor antagonists but exacerbated by agonists. DISCUSSION: We demonstrated the temporality of neuroendocrine-stress-mediated biological sequalae responsible for ozone-induced glucose metabolic dysfunction and mechanism in a rodent model. Stress hormones assessment with real-time glucose monitoring may be useful in identifying interactions among irritant pollutants and stress-related illnesses. https://doi.org/10.1289/EHP11088.

The utility of alternative models in particulate matter air pollution toxicology.

Exposure to particulate matter (PM) air pollution increases risk of adverse human health effects. As more attention is brought to bear on the problem of PM, traditional mammalian in vivo models struggle to keep up with the risk assessment challenges posed by the countless number of unique PM samples across air sheds with limited or no toxicity information. This review examines the utility of three higher throughput, alternative, in vivo animal models in PM toxicity research: Danio rerio (zebrafish), Caenorhabditis elegans (nematode), and Drosophila melanogaster (fruit fly). These model organisms vary in basic biology, ease of handling, methods of exposure to PM, number and types of available assays, and the degree to which they mirror human biology and responsiveness, among other differences. The use of these models in PM research dates back over a decade, with assessments of the toxicity of various PM sources including traffic-related combustion emissions, wildland fire smoke, and coal fly ash. This article reviews the use of these alternative model organisms in PM toxicity studies, their biology, the various assays developed, endpoints measured, their strengths and limitations, as well as their potential role in PM toxicity assessment and mechanistic research going forward.

Key Characteristics of Cardiovascular Toxicants.

BACKGROUND: The concept of chemical agents having properties that confer potential hazard called key characteristics (KCs) was first developed to identify carcinogenic hazards. Identification of KCs of cardiovascular (CV) toxicants could facilitate the systematic assessment of CV hazards and understanding of assay and data gaps associated with current approaches. OBJECTIVES: We sought to develop a consensus-based synthesis of scientific evidence on the KCs of chemical and nonchemical agents known to cause CV toxicity along with methods to measure them. METHODS: An expert working group was convened to discuss mechanisms associated with CV toxicity. RESULTS: The group identified 12 KCs of CV toxicants, defined as exogenous agents that adversely interfere with function of the CV system. The KCs were organized into those primarily affecting cardiac tissue (numbers 1-4 below), the vascular system (5-7), or both (8-12), as follows: 1) impairs regulation of cardiac excitability, 2) impairs cardiac contractility and relaxation, 3) induces cardiomyocyte injury and death, 4) induces proliferation of valve stroma, 5) impacts endothelial and vascular function, 6) alters hemostasis, 7) causes dyslipidemia, 8) impairs mitochondrial function, 9) modifies autonomic nervous system activity, 10) induces oxidative stress, 11) causes inflammation, and 12) alters hormone signaling. DISCUSSION: These 12 KCs can be used to help identify pharmaceuticals and environmental pollutants as CV toxicants, as well as to better understand the mechanistic underpinnings of their toxicity. For example, evidence exists that fine particulate matter [PM ≤2.5µm in aerodynamic diameter (PM2.5)] air pollution, arsenic, anthracycline drugs, and other exogenous chemicals possess one or more of the described KCs. In conclusion, the KCs could be used to identify potential CV toxicants and to define a set of test methods to evaluate CV toxicity in a more comprehensive and standardized manner than current approaches. https://doi.org/10.1289/EHP9321.

Exposure to Intermittent Noise Exacerbates the Cardiovascular Response of Wistar-Kyoto Rats to Ozone Inhalation and Arrhythmogenic Challenge.

Noise has become a prevalent public health problem across the world. Although there is a significant amount of data demonstrating the harmful effects of noise on the body, very little is known about how it impacts subsequent responses to other environmental stressors like air pollution, which tend to colocalize in urban centers. Therefore, this study was conducted to determine the effect of intermittent noise on cardiovascular function and subsequent responses to ozone (O3). Male Wistar-Kyoto rats implanted with radiotelemeters to non-invasively measure heart rate (HR) and blood pressure (BP), and assess heart rate variability (HRV) and baroreflex sensitivity (BRS) were kept in the quiet or exposed to intermittent white noise (85-90 dB) for one week and then exposed to either O3 (0.8 ppm) or filtered air. Left ventricular function and arrhythmia sensitivity were measured 24 h after exposure. Intermittent noise caused an initial increase in HR and BP, which decreased significantly later in the regimen and coincided with an increase in HRV and BRS. Noise caused HR and BP to be significantly elevated early during O3 and lower at the end when compared to animals kept in the quiet while the increased HRV and BRS persisted during the 24 h after. Lastly, noise increased arrhythmogenesis and may predispose the heart to mechanical function changes after O3. This is the first study to demonstrate that intermittent noise worsens the cardiovascular response to inhaled O3. These effects may occur due to autonomic changes and dysregulation of homeostatic controls, which persist one day after exposure to noise. Hence, co-exposure to noise should be taken into account when assessing the health effects of urban air pollution.

Early-life persistent vitamin D deficiency-induced cardiovascular dysfunction in mice is mediated by transient receptor potential C channels.

BACKGROUND: Studies indicate that chronic vitamin D deficiency (VDD) may predispose to hypertension, yet, there is very little data characterizing its direct cardiac effects. Vitamin D modulates the function of transient receptor potential C cation channels (TRPC), which is a mechanosensitive cation channel that plays a role in cardiac slow-force responses to hemodynamic changes. The purpose of this study was to determine the cardiac effects of VDD and the potential role of TRPC. METHODS: Three-week old mice were placed on a VDD or normal diet (ND) for 19 weeks. Mice were then implanted with radiotelemeters for the measurement of heart rate (HR) and heart rate variability (HRV), while a separate group was anesthetized to measure blood pressure (BP) and left ventricular function using an intraventricular probe. Animals were treated with a TRPC antagonist or vehicle after which they were challenged with dobutamine to measure cardiac responses. RESULTS: VDD mice had significantly increased BP (72 ± 3 mmHg vs. 62 ± 2 mmHg) and left ventricular pressure (LVP) (84.6 ± 0.8 mmHg vs. 78.2 ± 2.0 mmHg), and decreased cardiac contractility (-3 % vs. + 11 %) and HR response (+8 % vs. + 13 %) to dobutamine when compared to ND. These responses were blocked by the TRPC antagonist. HRV decreased with increasing dobutamine doses in ND but not VDD mice, however, the antagonist had no effect. CONCLUSION: VDD increases BP and alters cardiac mechanical function in mice, the latter appears to be mediated by TRPC, in particular TRPC6. Although the cardiac effects might be due to increased BP, it is likely that VDD also affects the function of the heart directly. This is the first study to demonstrate the potentially deleterious effects of VDD on cardiac function and the role of TRPC6 in this response.

Peat smoke inhalation alters blood pressure, baroreflex sensitivity, and cardiac arrhythmia risk in rats.

Wildland fires (WF) are linked to adverse health impacts related to poor air quality. The cardiovascular impacts of emissions from specific biomass sources are however unknown. The purpose of this study was to assess the cardiovascular impacts of a single exposure to peat smoke, a key regional WF air pollution source, and relate these to baroreceptor sensitivity and inflammation. Three-month-old male Wistar-Kyoto rats, implanted with radiotelemeters for continuous monitoring of heart rate (HR), blood pressure (BP), and spontaneous baroreflex sensitivity (BRS), were exposed once, for 1-hr, to filtered air or low (0.38 mg/m3 PM) or high (4.04 mg/m3) concentrations of peat smoke. Systemic markers of inflammation and sensitivity to aconitine-induced cardiac arrhythmias, a measure of latent myocardial vulnerability, were assessed in separate cohorts of rats 24 hr after exposure. PM size (low peat = 0.4-0.5 microns vs. high peat = 0.8-1.2 microns) and proportion of organic carbon (low peat = 77% vs. high peat = 65%) varied with exposure level. Exposure to high peat and to a lesser extent low peat increased systolic and diastolic BP relative to filtered air. In contrast, only exposure to low peat elevated BRS and aconitine-induced arrhythmogenesis relative to filtered air and increased circulating levels of low-density lipoprotein cholesterol, complement components C3 and C4, angiotensin-converting enzyme (ACE), and white blood cells. Taken together, exposure to peat smoke produced overt and latent cardiovascular consequences that were likely influenced by physicochemical characteristics of the smoke and associated adaptive homeostatic mechanisms.

A single exposure to eucalyptus smoke sensitizes rats to the postprandial cardiovascular effects of a high carbohydrate oral load.

OBJECTIVE: Previous studies have shown that air pollution exposure primes the body to heightened responses to everyday stressors of the cardiovascular system. The purpose of this study was to examine the utility of postprandial responses to a high carbohydrate oral load, a cardiometabolic stressor long used to predict cardiovascular risk, in assessing the impacts of exposure to eucalyptus smoke (ES), a contributor to wildland fire air pollution in the Western coast of the United States. MATERIALS AND METHODS: Three-month-old male Sprague Dawley rats were exposed once (1 h) to filtered air (FA) or ES (700 µg/m3 fine particulate matter), generated by burning eucalyptus in a tube furnace. Rats were then fasted for six hours the following morning, and subsequently administered an oral gavage of either water or a HC suspension (70 kcal% from carbohydrate), mimicking a HC meal. Two hours post gavage, cardiovascular ultrasound, cardiac pressure-volume (PV), and baroreceptor sensitivity assessments were made, and pulmonary and systemic markers assessed. RESULTS: ES inhalation alone increased serum interleukin (IL)-4 and nasal airway levels of gamma glutamyl transferase. HC gavage alone increased blood glucose, blood pressure, and serum IL-6 and IL-13 compared to water vehicle. By contrast, only ES-exposed and HC-challenged animals had increased PV loop measures of cardiac output, ejection fraction %, dP/dtmax, dP/dtmin, and stroke work compared to ES exposure alone and/or HC challenge alone. DISCUSSION AND CONCLUSIONS: Exposure to a model wildfire air pollution source modifies cardiovascular responses to HC challenge, suggesting air pollution sensitizes the body to systemic triggers.

Fetal growth outcomes following peri-implantation exposure of Long-Evans rats to noise and ozone differ by sex.

BACKGROUND: Exposure to air pollution and high levels of noise have both been independently associated with the development of adverse pregnancy outcomes including low birth weight. However, exposure to such environmental stressors rarely occurs in isolation and is often co-localized, especially in large urban areas. METHODS: The purpose of this study was to compare the effects of combined exposure to noise (N) or ozone (O3), compared to either exposure alone. Long-Evans dams were exposed to air or 0.4 ppm ozone for 4 h on gestation day (GD) 5 and 6, coinciding with implantation receptivity. A subset of dams from each exposure group was further exposed to intermittent white noise (~ 85 dB) throughout the dark cycle following each inhalation exposure (n = 14 - 16/group). Uterine artery ultrasound was performed on GD 15 and 21. Fetal growth characteristics and indicators of placental nutrient status were measured at GD 21. RESULTS: Exposure to ozone + quiet (O3 + Q) conditions reduced uterine arterial resistance at GD 15 compared to air + quiet (A + Q) exposure, with no further reduction by GD 21. By contrast, exposure to air + noise (A + N) significantly increased uterine arterial resistance at both GD 15 and 21. Notably, while peri-implantation exposure to O3 + Q conditions reduced male fetal weight at GD 21, this effect was not observed in the air + noise (A + N) or the ozone + noise (O3 + N) exposure groups. Fetal weight in female offspring was not reduced by ozone exposure alone (O3 + Q), nor was it affected by air + noise (A + N) or by combined ozone + noise (O3 + N) exposure. CONCLUSIONS: These data indicate that exposure to ozone and noise differentially impact uterine blood flow, particularly at mid-gestation, with only ozone exposure being associated with sex-dependent fetal growth retardation in male offspring.

Early Proteome Shift and Serum Bioactivity Precede Diesel Exhaust-induced Impairment of Cardiovascular Recovery in Spontaneously Hypertensive Rats.

Single circulating factors are often investigated to explain air pollution-induced cardiovascular dysfunction, yet broader examinations of the identity and bioactivity of the entire circulating milieu remain understudied. The purpose of this study was to determine if exposure-induced cardiovascular dysfunction can be coupled with alterations in both serum bioactivity and the circulating proteome. Two cohorts of Spontaneously Hypertensive Rats (SHRs) were exposed to 150 or 500 µg/m3 diesel exhaust (DE) or filtered air (FA). In Cohort 1, we collected serum 1 hour after exposure for proteomics analysis and bioactivity measurements in rat aortic endothelial cells (RAECs). In Cohort 2, we assessed left ventricular pressure (LVP) during stimulation and recovery from the sympathomimetic dobutamine HCl, one day after exposure. Serum from DE-exposed rats had significant changes in 66 serum proteins and caused decreased NOS activity and increased VCAM-1 expression in RAECs. While rats exposed to DE demonstrated increased heart rate at the start of LVP assessments, heart rate, systolic pressure, and double product fell below baseline in DE-exposed rats compared to FA during recovery from dobutamine, indicating dysregulation of post-exertional cardiovascular function. Taken together, a complex and bioactive circulating milieu may underlie air pollution-induced cardiovascular dysfunction.

High-Throughput Video Processing of Heart Rate Responses in Multiple Wild-type Embryonic Zebrafish per Imaging Field.

Heart rate assays in wild-type zebrafish embryos have been limited to analysis of one embryo per video/imaging field. Here we present for the first time a platform for high-throughput derivation of heart rate from multiple zebrafish (Danio rerio) embryos per imaging field, which is capable of quickly processing thousands of videos and ideal for multi-well platforms with multiple fish/well. This approach relies on use of 2-day post fertilization wild-type embryos, and uses only bright-field imaging, circumventing requirement for anesthesia or restraint, costly software/hardware, or fluorescently-labeled animals. Our original scripts (1) locate the heart and record pixel intensity fluctuations generated by each cardiac cycle using a robust image processing routine, and (2) process intensity data to derive heart rate. To demonstrate assay utility, we exposed embryos to the drugs epinephrine and clonidine, which increased or decreased heart rate, respectively. Exposure to organic extracts of air pollution-derived particulate matter, including diesel or biodiesel exhausts, or wood smoke, all complex environmental mixtures, decreased heart rate to varying degrees. Comparison against an established lower-throughput method indicated robust assay fidelity. As all code and executable files are publicly available, this approach may expedite cardiotoxicity screening of compounds as diverse as small molecule drugs and complex chemical mixtures.

Ozone Exposure During Implantation Increases Serum Bioactivity in HTR-8/SVneo Trophoblasts.

Implantation is a sensitive window in reproductive development during which disruptions may increase the risk of adverse pregnancy outcomes including intrauterine growth restriction. Ozone exposure during implantation in rats reduces fetal weight near the end of gestation, potentially though impaired trophoblast migration and invasion and altered implantation. The current study characterized changes in ventilation, pulmonary injury, and circulating factors including hormonal, inflammatory, and metabolic markers related to exposure to ozone (0.4-1.2 ppm) for 4-h on gestation days 5 and 6 (window of implantation) in Long-Evans dams. To determine the effects of this exposure on trophoblast function, placental-derived, first trimester, HTR-8/SVneo cells were exposed to serum from air- or ozone (0.8 ppm×4 h)-exposed dams and examined for impacts on metabolic capacity, wound-closure, and invasion. Peri-implantation exposure to ozone induced ventilatory dysfunction and lung vascular leakage in pregnant rats, with little effect on most of the circulating markers measured. However, ozone inhalation induced a significant reduction in several serum cytokines (interferon-γ, interleukin-6, and interleukin-13). Treatment of HTR-8/SVneo trophoblasts with serum from ozone-exposed dams for 16-h downregulated metabolic capacity, wound-closure, and invasion through a Matrigel membrane compared with both air-serum and fetal bovine serum-treated cells. Ozone-serum treated cells increased the release of a critical inhibitor of invasion and angiogenesis (soluble fms-like receptor 1; sFlt1) compared with air-serum treatment. Together, our data suggest that circulating factors in the serum of pregnant rats exposed to ozone during implantation receptivity can hinder critical processes of implantation (eg, invasion and migration) and impair trophoblast metabolic capacity.

Ambient Particulate Matter and Acrolein Co-Exposure Increases Myocardial Dyssynchrony in Mice via TRPA1.

Air pollution is a complex mixture of particulate matter and gases linked to adverse clinical outcomes. As such, studying responses to individual pollutants does not account for the potential biological responses resulting from the interaction of various constituents within an ambient air shed. We previously reported that exposure to high levels of the gaseous pollutant acrolein perturbs myocardial synchrony. Here, we examined the effects of repeated, intermittent co-exposure to low levels of concentrated ambient particulates (CAPs) and acrolein on myocardial synchrony and the role of transient receptor potential cation channel A1 (TRPA1), which we previously linked to air pollution-induced sensitization to triggered cardiac arrhythmia. Female B6129 and Trpa1-/- mice (n = 6/group) were exposed to filtered air (FA), CAPs (46 µg/m3 of PM2.5), Acrolein (0.42 ppm), or CAPs+Acrolein for 3 h/day, 2 days/week for 4 weeks. Cardiac ultrasound was conducted to assess cardiac synchronicity and function before and after the first exposure and after the final exposure. Heart rate variability (HRV), an indicator of autonomic tone, was assessed after the final exposure. Strain delay (time between peak strain in adjacent cardiac wall segments), an index of myocardial dyssynchrony, increased by 5-fold after the final CAPs+Acrolein exposure in B6129 mice compared with FA, CAPs, or Acrolein-exposed B6129 mice, and CAPs+Acrolein-exposed Trpa1-/- mice. Only exposure to acrolein alone increased the HRV high frequency domain (5-fold) in B6129 mice, but not in Trpa1-/- mice. Thus, repeated inhalation of pollutant mixtures may increase risk for cardiac responses compared with single or multiple exposures to individual pollutants through TRPA1 activation.

Aspirin pre-treatment modulates ozone-induced fetal growth restriction and alterations in uterine blood flow in rats.

Prenatal exposure to ozone has been linked to low birth weight in people and fetal growth restriction in rats. Clinical recommendations suggest use of low dose aspirin to lower risk of preeclampsia and intrauterine growth restriction in high-risk pregnancies, yet its utility in mitigating the postnatal effects of gestational ozone exposure is unknown. The present study investigated the possibility of low dose aspirin to mitigate the effects of ozone exposure during pregnancy. Exposure to ozone impaired uterine arterial flow and induced growth restriction in fetuses of both sexes. Aspirin treatment induced marginal improvements in ozone-induced uterine blood flow impairment. However, this resulted in a protection of fetal weight in dams given aspirin only in early pregnancy. Aspirin administration for the entirety of gestation increased placental weight and reduced antioxidant status, suggesting that prolonged exposure to low dose aspirin may induce placental inefficiency in our model of growth restriction.

Acute peat smoke inhalation sensitizes rats to the postprandial cardiometabolic effects of a high fat oral load.

Wildland fire emissions cause adverse cardiopulmonary outcomes, yet controlled exposure studies to characterize health impacts of specific biomass sources have been complicated by the often latent effects of air pollution. The aim of this study was to determine if postprandial responses after a high fat challenge, long used clinically to predict cardiovascular risk, would unmask latent cardiometabolic responses in rats exposed to peat smoke, a key wildland fire air pollution source. Male Wistar Kyoto rats were exposed once (1 h) to filtered air (FA), or low (0.36 mg/m3 particulate matter) or high concentrations (3.30 mg/m3) of peat smoke, generated by burning peat from an Irish bog. Rats were then fasted overnight, and then administered an oral gavage of a HF suspension (60 kcal% from fat), mimicking a HF meal, 24 h post-exposure. In one cohort, cardiac and superior mesenteric artery function were assessed using high frequency ultrasound 2 h post gavage. In a second cohort, circulating lipids and hormones, pulmonary and systemic inflammatory markers, and circulating monocyte phenotype using flow cytometry were assessed before or 2 or 6 h after gavage. HF gavage alone elicited increases in circulating lipids characteristic of postprandial responses to a HF meal. Few effects were evident after peat exposure in un-gavaged rats. By contrast, exposure to low or high peat caused several changes relative to FA-exposed rats 2 and 6 h post HF gavage including increased heart isovolumic relaxation time, decreased serum glucose and insulin, increased CD11 b/c-expressing blood monocytes, increased serum total cholesterol, alpha-1 acid glycoprotein, and alpha-2 macroglobulin (p = 0.063), decreased serum corticosterone, and increased lung gamma-glutamyl transferase. In summary, these findings demonstrate that a HF challenge reveals effects of air pollution that may otherwise be imperceptible, particularly at low exposure levels, and suggest exposure may sensitize the body to mild inflammatory triggers.