Annette C. Rohr

Generation and Quantification of Ultrafine Particles through Terpene/Ozone Reaction in a Chamber Setting

Terpene/ozone reactions produce gas- and condensed-phase products and thus contribute to both indoor and outdoor aerosol. These reactions may be important in indoor settings, where terpenes are generated from indoor sources and ambient ozone can reach significant levels. Moreover, airway irritation has been observed in mice exposed to terpene oxidation products (OPs). The aim of this study was to characterize a system for generating and quantifying ultrafine particles formed through terpene/ozone reactions in preparation for inhalation toxicology experiments. Two common monoterpenes, f -pinene and d -limonene, and a hemiterpene, isoprene, were investigated. Ozone and gas-phase terpene were introduced continuously into a reaction flow tube, from which reaction products entered a plexiglass chamber. Particle number, mass, and size distribution (∼15-750 nm) were monitored in the chamber for various reactant concentrations and air exchange rates (AERs). In all experiments, ozone was the limiting reagent and the reaction rate was much more rapid than the AER. Particles formed rapidly and in high concentrations in the pinene and limonene systems. Particle formation was slower in the isoprene system and fewer particles were formed; moreover, particle diameters were smaller. In all 3 systems, progressive growth of particles was observed due to condensation and coagulation processes. The isoprene system displayed instability with respect to aerosol characteristics and did not reach steady-state conditions. In the pinene system, ozone concentration was a strong predictor of steady-state particle number and mass concentration and particle diameter. The particle number was greater at higher AERs, but particles were smaller. This study is the first to incorporate measurement of ultrafine particles formed from terpene/ozone reactions into a controlled exposure chamber setting. Following system characterization, we will conduct mouse exposures to further investigate the respiratory effects of gas- and particle-phase terpene OPs.

UPPER AIRWAY AND PULMONARY EFFECTS OF OXIDATION PRODUCTS OF (+)- α -PINENE, d -LIMONENE, AND ISOPRENE IN BALB/ c MICE

The oxidation products (OPs) of ozone and the unsaturated hydrocarbons d -limonene, (+)-α -pinene, and isoprene have previously been shown to cause upper airway irritation in mice during 30-min acute exposures. This study evaluated the effects of OPs and the hydrocarbons themselves on the upper airways, the conducting airways, and the lungs over a longer exposure period. The time course of development of effects and the reversibility of effects were investigated; in addition, we assessed possible exacerbation of sensory responses of the airways to the unreacted hydrocarbons. Respiratory parameters in male BALB/ c mice were monitored via head-out plethysmography. Exposures to OPs or hydrocarbons were for 60 min, followed by a 30-min challenge period with air or hydrocarbon, and a 15-min recovery period with air only. Experiments were also performed where limonene/ozone exposures were separated 6 h from the challenge period. Ozone concentration in the reaction mixture was 3.4 ppm, and concentrations of hydrocarbons were 47 ppm (α -pinene), 51 ppm (d -limonene), and 465 ppm (isoprene). Due to reaction, the ozone concentration at the point of exposure was less than 0.35 ppm; exposure to 0.30 ppm ozone for 60 min did not produce effects different from air-exposed control animals. As previously established, upper airway irritation was a prominent effect of OP exposure. In addition, over the longer exposure period we observed the development of airflow limitation that persisted for at least 45 min postexposure. All effects from limonene/ozone exposures were reversible within 6 h. Exposures to OPs did not cause enhanced upper airway irritation during challenge with the hydrocarbons, indicating that a 1-h exposure to OPs did not increase the sensitivity of the upper respiratory system. However, airflow limitation was exacerbated in animals exposed to d -limonene alone immediately following exposure to limonene OPs. These findings suggest that terpene/ozone reaction products may have moderate-lasting adverse effects on both the upper airways and pulmonary regions. This may be important in the context of the etiology or exacerbation of lower airway symptoms in office workers, or of occupational asthma in workers involved in industrial cleaning operations.

The health significance of gas- and particle-phase terpene oxidation products: A review

The reactions between terpenes and ozone (or other oxidants) produce a wide variety of both gas- and particle-phase products. Terpenes are biogenic volatile organic compounds (VOCs) that are also contained in many consumer products. Ozone is present indoors since it infiltrates into the indoor environment and is emitted by some office and consumer equipment. Some of the gaseous products formed are irritating to biological tissues, while the condensed-phase products have received attention due to their contribution to ambient fine particulate matter (PM2.5) and its respective health significance. Despite common scientific questions, the indoor and ambient air research communities have tended to operate in isolation regarding this topic. This review critically evaluates the literature related to terpene oxidation products and attempts to synthesize results of indoor and ambient air studies to better understand the health significance of these materials and identify knowledge gaps. The review documents the results of a literature search covering terpene oxidation chemistry, epidemiological, toxicological, and controlled human exposure studies, as well as health studies focused more generically on secondary organic aerosol (SOA). The literature shows a clear role for gas-phase terpene oxidation products in adverse airway effects at high concentrations; however, whether these effects occur at more environmentally relevant levels is unclear. The evidence for toxicity of particle-phase products is less conclusive. Knowledge gaps and future research needs are outlined, and include the need for more consistency in study designs, incorporation of reaction product measurements into epidemiological studies conducted in both indoor and ambient settings, and more focused research on the toxicity of SOA, especially SOA of biogenic origin.

A comparison of vascular effects from complex and individual air pollutants indicates a role for monoxide gases and volatile hydrocarbons.

Background Emerging evidence suggests that the systemic vasculature may be a target of inhaled pollutants of vehicular origin. We have identified several murine markers of vascular toxicity that appear sensitive to inhalation exposures to combustion emissions. Objective We sought to examine the relative impact of various pollutant atmospheres and specific individual components on these markers of altered vascular transcription and lipid peroxidation. Methods Apolipoprotein E knockout (ApoE−/−) mice were exposed to whole combustion emissions (gasoline, diesel, coal, hardwood), biogenically derived secondary organic aerosols (SOAs), or prominent combustion-source gases [nitric oxide (NO), NO2, carbon monoxide (CO)] for 6 hr/day for 7 days. Aortas were assayed for transcriptional alterations of endothelin-1 (ET-1), matrix metalloproteinase-9 (MMP-9), tissue inhibitor of metalloproteinase-2 (TIMP-2), and heme oxygenase-1 (HO-1), along with measures of vascular lipid peroxides (LPOs) and gelatinase activity. Results We noted transcriptional alterations with exposures to gasoline and diesel emissions. Interestingly, ET-1 and MMP-9 transcriptional effects could be recreated by exposure to CO and NO, but not NO2 or SOAs. Gelatinase activity aligned with levels of volatile hydrocarbons and also monoxide gases. Neither gases nor particles induced vascular LPO despite potent effects from whole vehicular emissions. Conclusions In this head-to-head comparison of the effects of several pollutants and pollutant mixtures, we found an important contribution to vascular toxicity from readily bioavailable monoxide gases and possibly from volatile hydrocarbons. These data support a role for traffic-related pollutants in driving cardiopulmonary morbidity and mortality.

Comparative acute lung inflammation induced by atmospheric PM and size-fractionated tire particles.

A comparison of the effects produced by size-fractionated tire particles (TP10 and TP2.5) and similar-sized urban particulate matter (PM10 and PM2.5), collected in Milan in 2007, on the lungs of mice has been performed. The focus is on early acute lung responses following intratracheal instillation of aerosolized particles at a 3-h recovery period. Together with bronchoalveolar lavage (BAL) conventional endpoints like total and differential cell counts, total protein, alkaline phosphatase, lactate dehydrogenase and pro-inflammatory cytokines (TNF-alpha, MIP-2), the expression of different stress protein markers (caspase8, Hsp70, H0-1, NF-kB) was evaluated 3h after particle instillation into Balb/c mice. The TP2.5 fraction reached the alveolar spaces and produced an acute inflammatory response as evidenced by increased LDH and AP activities, total protein and Hsp70 content. TNF-alpha and MIP-2 production was significantly increased and polymorphonuclear neutrophils (PMN) recruitment was apparent. The TP10 fraction distributed mainly in the bronchial district and the only modified BAL parameter was the expression of MIP-2. PM2.5 induced an inflammatory response lesser in magnitude than that produced by PM10 fraction. The TNF-alpha increase was not significant, and HO-1, though significantly increased with respect to the control, was unable to reduce NF-kB activation, suggesting a role of the endotoxin component of PM in stimulating a pro-inflammatory limited response. This response was maximized by the PM10 that induced a significant increase in MIP-2, TNF-alpha, and HO-1. Lung immunohistochemistry showed fine particles, TPs in particular, being able to deeply penetrate and rapidly induce inflammatory events in the parenchyma, even involving endothelial cells, while PM10 produced a strong pro-inflammatory response mediated by the bronchiolar cells and residential macrophages of the proximal alveolar sacs, likely as a consequence of its larger dimension and endotoxin content. These results provide evidence of variable inflammatory mechanisms in mouse lungs in response to both urban PM and tire particles.

Lung toxicity induced by intratracheal instillation of size-fractionated tire particles

Tire particles (TP) represent a significant component of urban air pollution (PM), constituting more than 10% of PM10 mass at urban locations with heavy traffic. The purpose of this study was to evaluate the effects of size-fractionated TP in an animal exposure model frequently used to assess the health effects of air pollutants. Potential pro-inflammatory and toxic effects of TP2.5 (<2.5 microm) and TP10 (<10 microm) were investigated through instillation of suspensions of these materials in BALB/c mice. Bronchoalveolar lavage fluid (BALF) was screened for total protein, lactate dehydrogenase (LDH), alkaline phosphatase (AP), and beta-glucuronidase (B-Gluc) as markers of cytotoxicity; glutathione (GSH) and superoxide dismutase (SOD) as markers of oxidative potential; and tumor necrosis factor-alpha (TNF-alpha), macrophage inflammatory protein-2 (MIP-2), and inflammatory cells as markers of inflammation. Concomitantly, histological analysis of TP-exposed lungs was performed. A single intratracheal instillation of 10 microg/100 microl, 100 microg/100 microl or 200 microg/100 microl was performed, and after 24h mice were euthanized and BALF examined. Inflammatory cellular profiles showed dose-dependent responses after TP10 exposure, while strong cytotoxic effects, including increases in total protein, LDH and AP, were observed to be associated to TP2.5 exposure. Histologically, TP10-treated lungs mainly showed inflammatory tissue infiltration, in contrast to TP2.5-treated lungs, where lysis of the alveolar barrier appeared to be the most characteristic lesion. Our biochemical, cytological, and histological results indicated differential lung toxicity mechanisms elicited by size-fractionated TP, in agreement with other studies performed in in vivo systems that have shown that lung responses to inhaled or instilled particles are affected by particle size. We conclude that lung toxicity induced by TP10 was primarily due to macrophage-mediated inflammatory events, while toxicity induced by TP2.5 appeared to be related more closely to cytotoxicity.

Toxicological evaluation of realistic emission source aerosols (TERESA): summary and conclusions

The toxicological evaluation of realistic emissions of source aerosols (TERESA) study seeks to delineate health effects of aerosols formed from emissions of particulate matter sources. This series of papers reports the findings of experiments using coal-fired power plants as the source of emissions and this paper summarizes the findings and knowledge acquired from these studies. Emissions were drawn directly from the stacks of three coal-fired power plants in the US, and photochemically aged in a mobile laboratory to simulate downwind power plant plume processing. The power plants used different sources of coal and had different emission controls. Exposure scenarios included primary particles, secondary particles and mixtures of these with common atmospheric constituents (α-pinene and ammonia). Extensive exposure characterization was carried out, and toxicological outcomes were evaluated in Sprague-Dawley rats exposed to different emission scenarios. Breathing pattern, pulmonary inflammatory responses, in vivo pulmonary and cardiac chemiluminescence and cardiac response in a model of acute myocardial infarction were assessed. The results showed no response or relatively mild responses to the inhaled aerosols studied; complex scenarios which included oxidized emissions and α-pinene to simulate biogenic secondary organic aerosol tended to induce more statistically significant responses than scenarios of oxidized and non-oxidized emissions alone. Relating adverse effects to specific components did not consistently identify a toxic constituent. These findings are consistent with most of the previously published studies using pure compounds to model secondary power plant emissions, but importantly add substantial complexity and thus have considerable merit in defining toxicological responses.

The effects of PM 2.5 and its components from indoor and outdoor sources on cough and wheeze symptoms in asthmatic children

Particulate matter with aerodynamic diameter <2.5 μm (PM2.5) is associated with asthma exacerbation. In the Children’s Air Pollution Asthma Study, we investigated the longitudinal association of PM2.5 and its components from indoor and outdoor sources with cough and wheeze symptoms in 36 asthmatic children. The sulfur tracer method was used to estimate infiltration factors. Mixed proportional odds models for an ordinal response were used to relate daily cough and wheeze scores to PM2.5 exposures. The odds ratio associated with being above a given symptom score for a SD increase in PM2.5 from indoor sources (PMIS) was 1.24 (95% confidence interval: 0.92–1.68) for cough and 1.63 (1.11–2.39) for wheeze. Ozone was associated with wheeze (1.82, 1.19–2.80), and cough was associated with indoor PM2.5 components from outdoor sources (denoted with subscript “OS”) bromine (BrOS: 1.32, 1.05–1.67), chlorine (ClOS: 1.27, 1.02–1.59) and pyrolyzed organic carbon (OPOS: 1.49, 1.12–1.99). The highest effects were seen in the winter for cough with sulfur (SOS: 2.28, 1.01–5.16) and wheeze with organic carbon fraction 2 (OC2OS: 7.46, 1.19–46.60). Our results indicate that exposure to components originating from outdoor sources of photochemistry, diesel and fuel oil combustion is associated with symptom’s exacerbation, especially in the winter. PM2.5 mass of indoor origin was more strongly associated with wheeze than with cough.

Sources of indoor air pollution in New York City residences of asthmatic children.

Individuals spend ∼90% of their time indoors in proximity to sources of particulate and gaseous air pollutants. The sulfur tracer method was used to separate indoor concentrations of particulate matter (PM) PM2.5 mass, elements and thermally resolved carbon fractions by origin in New York City residences of asthmatic children. Enrichment factors relative to sulfur concentrations were used to rank species according to the importance of their indoor sources. Mixed effects models were used to identify building characteristics and resident activities that contributed to observed concentrations. Significant indoor sources were detected for OC1, Cl, K and most remaining OC fractions. We attributed 46% of indoor PM2.5 mass to indoor sources related to OC generation indoors. These sources include cooking (NO2, Si, Cl, K, OC4 and OP), cleaning (most OC fractions), candle/incense burning (black carbon, BC) and smoking (K, OC1, OC3 and EC1). Outdoor sources accounted for 28% of indoor PM2.5 mass, mainly photochemical reaction products, metals and combustion products (EC, EC2, Br, Mn, Pb, Ni, Ti, V and S). Other indoor sources accounted for 26% and included re-suspension of crustal elements (Al, Zn, Fe, Si and Ca). Indoor sources accounted for ∼72% of PM2.5 mass and likely contributed to differences in the composition of indoor and outdoor PM2.5 exposures.

Cardiopulmonary response to inhalation of biogenic secondary organic aerosol

An irradiation chamber designed for reproducible generation of inhalation test atmospheres of secondary organic aerosol (SOA) was used to evaluate cardiopulmonary responses in rodents exposed to SOA derived from the oxidation of α-pinene. SOA atmospheres were produced with 10:1 ratios of α-pinene:nitrogen oxides (NOx) and 10:1:1 ratios of α-pinene:nitrogen oxides:sulfur dioxide (SO2). SOA atmospheres were produced to yield 200 μg m−3 of particulate matter (PM). Exposures were conducted downstream of honeycomb denuders employed to remove the gas-phase precursors and reaction products. Nose-only exposures were conducted with both rats (pulmonary effects) and mice (pulmonary and cardiovascular effects). Composition of the atmospheres was optimized to ensure that the SOA generated resembled SOA observed in previous irradiation studies, and contained specific SOA compounds of interest (e.g., organosulfates) identified in ambient air. Pulmonary and cardiovascular toxicity were measured in two different rodent species. In situ chemiluminescence and thiobarbituric acid– reactive substances (TBARS) were used to evaluate oxidative reactions in the F344 rats. ApoE−/− mice were exposed for 7 days and measurements of TBARS and gene expression of heme oxygenase-1 (HO-1), endothelin-1 (ET-1), matrix metalloproteinase-9 (MMP-9) were made in aorta. Pulmonary inflammatory responses in both species were measured by bronchoalveolar lavage fluid (BALF) cell counts. No pulmonary inflammation was observed in either species. A mild response was observed in mouse aorta for the upregulation of HO-1 and MMP-9, but was not seen for ET-1. Overall, α-pinene–derived SOA, including SOA that included organosulfate compounds, revealed limited biological response after short-term inhalation exposures