Soy biodiesel emissions have reduced inflammatory effects compared to diesel emissions in healthy and allergic mice.

Toxicity of exhaust from combustion of petroleum diesel (B0), soy-based biodiesel (B100), or a 20% biodiesel/80% petrodiesel mix (B20) was compared in healthy and house dust mite (HDM)-allergic mice. Fuel emissions were diluted to target fine particulate matter (PM(2.5)) concentrations of 50, 150, or 500 µg/m(3). Studies in healthy mice showed greater levels of neutrophils and MIP-2 in bronchoalveolar lavage (BAL) fluid 2 h after a single 4-h exposure to B0 compared with mice exposed to B20 or B100. No consistent differences in BAL cells and biochemistry, or hematological parameters, were observed after 5 d or 4 weeks of exposure to any of the emissions. Air-exposed HDM-allergic mice had significantly increased responsiveness to methacholine aerosol challenge compared with non-allergic mice. Exposure to any of the emissions for 4 weeks did not further increase responsiveness in either non-allergic or HDM-allergic mice, and few parameters of allergic inflammation in BAL fluid were altered. Lung and nasal pathology were not significantly different among B0-, B20-, or B100-exposed groups. In HDM-allergic mice, exposure to B0, but not B20 or B100, significantly increased resting peribronchiolar lymph node cell proliferation and production of T(H)2 cytokines (IL-4, IL-5, and IL-13) and IL-17 in comparison with air-exposed allergic mice. These results suggest that diesel exhaust at a relatively high concentration (500 µg/m(3)) can induce inflammation acutely in healthy mice and exacerbate some components of allergic responses, while comparable concentrations of B20 or B100 soy biodiesel fuels did not elicit responses different from those caused by air exposure alone.

Cardiopulmonary toxicity of peat wildfire particulate matter and the predictive utility of precision cut lung slices.

BACKGROUND: Emissions from a large peat fire in North Carolina in 2008 were associated with increased hospital admissions for asthma and the rate of heart failure in the exposed population. Peat fires often produce larger amounts of smoke and last longer than forest fires, however few studies have reported on their toxicity. Moreover, reliable alternatives to traditional animal toxicity testing are needed to reduce the number of animals required for hazard identification and risk assessments. METHODS: Size-fractionated particulate matter (PM; ultrafine, fine, and coarse) were obtained from the peat fire while smoldering (ENCF-1) or when nearly extinguished (ENCF-4). Extracted samples were analyzed for chemical constituents and endotoxin content. Female CD-1 mice were exposed via oropharyngeal aspiration to 100 µg/mouse, and assessed for relative changes in lung and systemic markers of injury and inflammation. At 24 h post-exposure, hearts were removed for ex vivo functional assessments and ischemic challenge. Lastly, 8 mm diameter lung slices from CD-1 mice were exposed (11 µg) ± co-treatment of PM with polymyxin B (PMB), an endotoxin-binding compound. RESULTS: On an equi-mass basis, coarse ENCF-1 PM had the highest endotoxin content and elicited the greatest pro-inflammatory responses in the mice including: increases in bronchoalveolar lavage fluid protein, cytokines (IL-6, TNF-α, and MIP-2), neutrophils and intracellular reactive oxygen species (ROS) production. Exposure to fine or ultrafine particles from either period failed to elicit significant lung or systemic effects. In contrast, mice exposed to ENCF-1 ultrafine PM developed significantly decreased cardiac function and greater post-ischemia-associated myocardial infarction. Finally, similar exposures to mouse lung slices induced comparable patterns of cytokine production; and these responses were significantly attenuated by PMB. CONCLUSIONS: The findings suggest that exposure to coarse PM collected during a peat fire causes greater lung inflammation in association with endotoxin and ROS, whereas the ultrafine PM preferentially affected cardiac responses. In addition, lung tissue slices were shown to be a predictive, alternative assay to assess pro-inflammatory effects of PM of differing size and composition. Importantly, these toxicological findings were consistent with the cardiopulmonary health effects noted in epidemiologic reports from exposed populations.

Comparative lung toxicity of engineered nanomaterials utilizing in vitro, ex vivo and in vivo approaches.

BACKGROUND: Although engineered nanomaterials (ENM) are currently regulated either in the context of a new chemical, or as a new use of an existing chemical, hazard assessment is still to a large extent reliant on information from historical toxicity studies of the parent compound, and may not take into account special properties related to the small size and high surface area of ENM. While it is important to properly screen and predict the potential toxicity of ENM, there is also concern that current toxicity tests will require even heavier use of experimental animals, and reliable alternatives should be developed and validated. Here we assessed the comparative respiratory toxicity of ENM in three different methods which employed in vivo, in vitro and ex vivo toxicity testing approaches. METHODS: Toxicity of five ENM (SiO2 (10), CeO2 (23), CeO2 (88), TiO2 (10), and TiO2 (200); parentheses indicate average ENM diameter in nm) were tested in this study. CD-1 mice were exposed to the ENM by oropharyngeal aspiration at a dose of 100 µg. Mouse lung tissue slices and alveolar macrophages were also exposed to the ENM at concentrations of 22-132 and 3.1-100 µg/mL, respectively. Biomarkers of lung injury and inflammation were assessed at 4 and/or 24 hr post-exposure. RESULTS: Small-sized ENM (SiO2 (10), CeO2 (23), but not TiO2 (10)) significantly elicited pro-inflammatory responses in mice (in vivo), suggesting that the observed toxicity in the lungs was dependent on size and chemical composition. Similarly, SiO2 (10) and/or CeO2 (23) were also more toxic in the lung tissue slices (ex vivo) and alveolar macrophages (in vitro) compared to other ENM. A similar pattern of inflammatory response (e.g., interleukin-6) was observed in both ex vivo and in vitro when a dose metric based on cell surface area (µg/cm(2)), but not culture medium volume (µg/mL) was employed. CONCLUSION: Exposure to ENM induced acute lung inflammatory effects in a size- and chemical composition-dependent manner. The cell culture and lung slice techniques provided similar profiles of effect and help bridge the gap in our understanding of in vivo, ex vivo, and in vitro toxicity outcomes.

Biological effects of desert dust in respiratory epithelial cells and a murine model.

As a result of the challenge of recent dust storms to public health, we tested the postulate that desert dust collected in the southwestern United States imparts a biological effect in respiratory epithelial cells and an animal model. Two samples of surface sediment were collected from separate dust sources in northeastern Arizona. Analysis of the PM20 fraction demonstrated that the majority of both dust samples were quartz and clay minerals (total SiO2 of 52 and 57%). Using respiratory epithelial and monocytic cell lines, the two desert dusts increased oxidant generation, measured by Amplex Red fluorescence, along with carbon black (a control particle), silica, and NIST 1649 (an ambient air pollution particle). Cell oxidant generation was greatest following exposures to silica and the desert dusts. Similarly, changes in RNA for superoxide dismutase-1, heme oxygenase-1, and cyclooxygenase-2 were also greatest after silica and the desert dusts supporting an oxidative stress after cell exposure. Silica, desert dusts, and the ambient air pollution particle NIST 1649 demonstrated a capacity to activate the p38 and ERK1/2 pathways and release pro-inflammatory mediators. Mice, instilled with the same particles, showed the greatest lavage concentrations of pro-inflammatory mediators, neutrophils, and lung injury following silica and desert dusts. We conclude that, comparable to other particles, desert dusts have a capacity to (1) influence oxidative stress and release of pro-inflammatory mediators in respiratory epithelial cells and (2) provoke an inflammatory injury in the lower respiratory tract of an animal model. The biological effects of desert dusts approximated those of silica.

Biomarkers of acute respiratory allergen exposure: screening for sensitization potential.

Effective hazard screening will require the development of high-throughput or in vitro assays for the identification of potential sensitizers. The goal of this preliminary study was to identify potential biomarkers that differentiate the response to allergens vs non-allergens following an acute exposure in naïve individuals. Female BALB/c mice received a single intratracheal aspiration exposure to Metarhizium anisopliae crude antigen (MACA) or bovine serum albumin (BSA) in Hank's Balanced Salt Solution (HBSS) or HBSS alone. Mice were terminated after 1, 3, 6, 12, 18 and 24 h. Bronchoalveolar lavage fluid (BALF) was evaluated to determine total and differential cellularity, total protein concentration and LDH activity. RNA was isolated from lung tissue for microarray analysis and qRT-PCR. MACA administration induced a rapid increase in BALF neutrophils, lymphocytes, eosinophils and total protein compared to BSA or HBSS. Microarray analysis demonstrated differential expression of genes involved in cytokine production, signaling, inflammatory cell recruitment, adhesion and activation in 3 and 12 h MACA-treated samples compared to BSA or HBSS. Further analyses allowed identification of approximately 100 candidate biomarker genes. Eleven genes were selected for further assessment by qRT-PCR. Of these, 6 demonstrated persistently increased expression (Ccl17, Ccl22, Ccl7, Cxcl10, Cxcl2, Saa1), while C3ar1 increased from 6-24 h. In conclusion, a single respiratory exposure of mice to an allergenic mold extract induces an inflammatory response which is distinct in phenotype and gene transcription from the response to a control protein. Further validation of these biomarkers with additional allergens and irritants is needed. These biomarkers may facilitate improvements in screening methods.

Role of oxidative stress on diesel-enhanced influenza infection in mice.

Numerous studies have shown that air pollutants, including diesel exhaust (DE), reduce host defenses, resulting in decreased resistance to respiratory infections. This study sought to determine if DE exposure could affect the severity of an ongoing influenza infection in mice, and examine if this could be modulated with antioxidants. BALB/c mice were treated by oropharyngeal aspiration with 50 plaque forming units of influenza A/HongKong/8/68 and immediately exposed to air or 0.5 mg/m3 DE (4 hrs/day, 14 days). Mice were necropsied on days 1, 4, 8 and 14 post-infection and lungs were assessed for virus titers, lung inflammation, immune cytokine expression and pulmonary responsiveness (PR) to inhaled methacholine. Exposure to DE during the course of infection caused an increase in viral titers at days 4 and 8 post-infection, which was associated with increased neutrophils and protein in the BAL, and an early increase in PR. Increased virus load was not caused by decreased interferon levels, since IFN-ß levels were enhanced in these mice. Expression and production of IL-4 was significantly increased on day 1 and 4 p.i. while expression of the Th1 cytokines, IFN-γ and IL-12p40 was decreased. Treatment with the antioxidant N-acetylcysteine did not affect diesel-enhanced virus titers but blocked the DE-induced changes in cytokine profiles and lung inflammation. We conclude that exposure to DE during an influenza infection polarizes the local immune responses to an IL-4 dominated profile in association with increased viral disease, and some aspects of this effect can be reversed with antioxidants.

Increased lung resistance after diesel particulate and ozone co-exposure not associated with enhanced lung inflammation in allergic mice.

Exposure to diesel exhaust particulate matter (DEP) exacerbates asthma. Likewise, similar effects have been reported with exposure to the oxidizing air pollutant ozone (O(3)). Since levels of both pollutants in ambient air tend to be simultaneously elevated, we investigated the possible synergistic effect of these agents on the exacerbation of allergic airways disease in mice. Male BALB/c mice were sensitized ip with ovalbumin (Ova) or vehicle only, then exposed once per week for 4 wk via nose-only inhalation (4 h) to the PM(2.5) fraction of DEP (2 mg/m(3)), O(3) (0.5 ppm), DEP and O(3), or filtered air, and then challenged with aerosolized ovalbumin. Ova sensitization in air-exposed mice enhanced pulmonary inflammatory cell infiltration, several indicators of injury in the lung (lactate dehydrogenase, albumin and total protein), and lung resistance (R(L)) and elastance (E(L)) in response to methacholine (MCh) aerosol challenge. DEP exposure did not enhance the Ova-induced increase in pulmonary cell infiltration, indicators of injury, or R(L) and E(L). O(3) exposure enhanced the Ova-induced increase in inflammatory cell infiltration and N-acetylglucosaminidase (NAG) in the lung, but had no effect on R(L) or E(L). DEP co-exposure significantly attenuated the O(3)-induced increase in cell infiltration and indicators of injury; co-exposure had no effect on E(L) relative to air-exposed Ova-sensitized mice. However, only DEP-O(3) co-exposure significantly increased the Ova-induced increase in R(L). Thus, O(3) and DEP co-exposure exacerbated airways hyperresponsiveness, a response that was not associated with parallel increases in pulmonary inflammation and one that may be mediated by a unique mechanism.

Th2 Cytokines in Skin Draining Lymph Nodes and Serum IgE Do Not Predict Airway Hypersensitivity to Intranasal Isocyanate Exposure in Mice.

Isocyanate exposure in the workplace has been linked to asthma and allergic rhinitis. Recently, investigators have proposed that Th2 cytokine responses in lymph nodes draining the site of dermal application of chemicals including isocyanates may be used to identify sensitizers that cause asthma-like responses. The purpose of this study was to determine if the cytokine profile induced after dermal sensitization with isocyanates and serum IgE predict immediate (IHS) and methacholine-induced late (LHS) respiratory hypersensitivity responses after intranasal challenge. Dermal application of hexylmethane diisocyanate (HMDI), toluene diisocyanate (TDI), or methylene diisocyanate (MDI) significantly increased interleukin-4 (IL-4), IL-5, and IL-13 secretion in parotid lymph node cells. Isophorone diisocyanate (IPDI) increased IL-4 and IL-13, but not IL-5. Tolyl(mono)isocyanate (TMI), tetramethylene xylene diisocyanate (TMXDI), or the contact sensitizer dinitrochlorobenzene (DNCB), only induced minor increases in some of the Th2 cytokines. HMDI, TDI, MDI, and IPDI elicited greater increases in total serum IgE than DNCB, TMI, and TMXDI. All chemicals except TMXDI caused IHS after intranasal challenge of sensitized female BALB/c mice. Only HMDI-, TMI-, or TMXDI-sensitized and challenged mice had increases in LHS. All chemicals elicited epithelial cytotoxicity indicative of nasal airway irritation. The discordance between dermal cytokine profiles and respiratory responses suggests that dermal responses do not necessarily predict respiratory responses. Serum IgE also was not predictive of the respiratory responses to the isocyanates, suggesting that other unknown mechanisms may be involved.

Comparative toxicity of size-fractionated airborne particulate matter obtained from different cities in the United States.

Hundreds of epidemiological studies have shown that exposure to ambient particulate matter (PM) is associated with dose-dependent increases in morbidity and mortality. While early reports focused on PM less than 10 microm (PM10), numerous studies have since shown that the effects can occur with PM stratified into ultrafine (UF), fine (FI), and coarse (CO) size modes despite the fact that these materials differ significantly in both evolution and chemistry. Furthermore the chemical makeup of these different size fractions can vary tremendously depending on location, meteorology, and source profile. For this reason, high-volume three-stage particle impactors with the capacity to collect UF, FI, and CO particles were deployed to four different locations in the United States (Seattle, WA; Salt Lake City, UT; Sterling Forest and South Bronx, NY), and weekly samples were collected for 1 mo in each place. The particles were extracted, assayed for a standardized battery of chemical components, and instilled into mouse lungs (female BALB/c) at doses of 25 and 100 microg. Eighteen hours later animals were euthanized and parameters of injury and inflammation were monitored in the bronchoalveolar lavage fluid and plasma. Of the four locations, the South Bronx coarse fraction was the most potent sample in both pulmonary and systemic biomarkers, with a strong increase in lung inflammatory cells as well as elevated levels of creatine kinase in the plasma. These effects did not correlate with lipopolysaccharide (LPS) or total zinc or sulfate content, but were associated with total iron. Receptor source modeling on the PM2.5 samples showed that the South Bronx sample was heavily influenced by emissions from coal fired power plants (31%) and mobile sources (22%). Further studies will assess how source profiles correlate with the observed effects for all locations and size fractions.

Inconsistencies between cytokine profiles, antibody responses, and respiratory hyperresponsiveness following dermal exposure to isocyanates.

Cytokine profiling of local lymph node responses has been proposed as a simple test to identify chemicals, such as low molecular weight diisocyanates, that pose a significant risk of occupational asthma. Previously, we reported cytokine messenger RNA (mRNA) profiles for dinitrochlorobenzene (DNCB) and six isocyanates: toluene diisocyanate, diphenylmethane-4,4'-diisocyanate, dicyclohexylmethane-4,4'-diisocyanate, isophorone diisocyanate, p-tolyl(mono)isocyanate, and meta-tetramethylene xylene diisocyanate. The present study was conducted to test the hypothesis that relative differences in the cytokine profile are predictive of relative differences in total serum immunoglobulin (Ig) E and respiratory responses to methacholine (Mch) following dermal exposure to the chemicals. After a preliminary experiment to determine an exposure regimen sufficient to achieve responses to Mch following dermal diisocyanate exposure, BALB/c mice received nine dermal exposures over a period of 28 days to one of six isocyanates, DNCB, or vehicle. Mice were then challenged with increasing doses of Mch and responsiveness was assessed using whole-body plethysmography. Serum antibody responses and cytokine mRNA profiles in the draining lymph node were also assessed. In separate experiments, cytokine protein assays were performed after five dermal exposures over a 14-day period. The response pattern for interleukin (IL)-4, IL-10, and IL-13 for the different isocyanates was highly reproducible as determined by RNAse protection assay, reverse transcription-PCR, or cytokine protein levels. However, the relative differences in T-helper cytokine profiles were not predictive of relative differences in either total serum IgE or respiratory responses to Mch following dermal exposure. The data suggest that the cytokine profiling approach needs to be further developed and refined before adoption and that other approaches to hazard identification should be pursued as well. Based on the weight of evidence from all the assays performed, it appears that all six isocyanates tested have some potential to cause respiratory hypersensitivity following dermal exposure.

A murine model for low molecular weight chemicals: differentiation of respiratory sensitizers (TMA) from contact sensitizers (DNFB).

Exposure to low molecular weight (LMW) chemicals contributes to both dermal and respiratory sensitization and is an important occupational health problem. Our goal was to establish an in vivo murine model for hazard identification of LMW chemicals that have the potential to induce respiratory hypersensitivity (RH). We used a dermal sensitization protocol followed by a respiratory challenge with the evaluation of endpoints typically associated with RH in human disease. Trimellitic anhydride (TMA) was used as a prototype respiratory sensitizer and was compared to the dermal sensitizer; 2,4-dinitrofluorobenzene (DNFB), along with vehicle controls. BALB/c mice were dermally sensitized using two exposure protocols. Mice in both protocols were dermally exposed on experimental days; D-18 and D-17 (abdomen), and D-13 (ear). On D 0 mice received an intratracheal (IT) challenge. The mice in Protocol 2 were abdominally exposed twice with the addition of exposures on D-25 and D-24. Results indicate that mice required the additional dermal sensitization and the IT challenge (Protocol 2) to significantly elevate total IgE in serum and bronchoalveolar lavage fluid (BALF). Additional responses suggestive of RH were seen following Protocol 2, including increases in BALF cell numbers and neutrophils post IT with TMA (but not DNFB). These data suggest that the dermal sensitization and IT challenge followed by evaluation of serum antibodies and lung parameters are a reasonable and logistically feasible approach towards the development of a model for RH responses to LMW chemicals.

Ultraviolet radiation downregulates allergy in BALB/c mice.

The immunosuppressive effects of exposure to ultraviolet radiation (UVR) are well known and the underlying mechanisms extensively studied. The suppression of Th1 appears to account for UVR suppression of contact hypersensitivity and delayed-type hypersensitivity responses and increased susceptibility to certain infections and tumor development. The underlying mechanisms suggest Th2-mediated responses associated with immediate-type hypersensitivity and allergic lung disease should be unchanged or possibly enhanced by UVR. The hypothesis that UVR exposure enhances allergic lung disease in BALB/c mice was tested. Effects of UVR on sensitization and elicitation of respiratory hypersensitivity were assessed using a fungal extract, Metarhizium anisopliae (MACA), as the allergen. BALB/c mice were sham or UVR (8 KJ/m(2)) exposed 3d before involuntary aspiration (IA) of MACA or vehicle. The mice received UVR exposures before the first and second of three IAs in the sensitization protocol and 3 d before the fourth IA in the elicitation protocol. Serum and bronchoalveolar lavage fluid (BALF) were harvested before (d 21, sensitization/d 24, elicitation) and at 1 (d 22/d 28), 3 (d 24/d 29), and 7 (d 28/d 35) d following the last IA. UVR exposure prior to sensitization suppressed two hallmarks of allergic disease, immune-mediated inflammation (eosinophil influx) and total immunoglobulin (Ig)E compared to the sham-UVR controls. There were no differences attributable to UVR exposure in previously sensitized mice. These data suggest that UVR exposure prior to sensitization suppresses allergic responses but has no effect on the elicitation of allergic responses in previously sensitized individuals. Consequently, there is no evidence that exposure to UVR enhances the induction or expression of allergic lung disease.