Zebrafish irritant responses to wildland fire-related biomass smoke are influenced by fuel type, combustion phase, and byproduct chemistry.

Human exposure to wildfire-derived particulate matter (PM) is linked to adverse health outcomes; however, little is known regarding the influence of biomass fuel type and burn conditions on toxicity. The aim of this study was to assess the irritant potential of extractable organic material (EOM) of biomass smoke condensates from five fuels (eucalyptus, pine, pine needle, peat, or red oak), representing various fire-prone regions of the USA, burned at two temperatures each [flaming (approximately 640°C) or (smoldering approximately 500°C)] using a locomotor assay in zebrafish (Danio rerio) larvae. It was postulated that locomotor responses, as measures of irritant effects, might be dependent upon fuel type and burn conditions and that these differences relate to combustion byproduct chemistry. To test this, locomotor activity was tracked for 60 min in 6-day-old zebrafish larvae (25-32/group) immediately after exposure to 0.4% dimethyl sulfoxide (DMSO) vehicle or EOM from the biomass smoke condensates (0.3-30 µg EOM/ml; half-log intervals). All EOM samples produced concentration-dependent irritant responses. Linear regression analysis to derive rank-order potency indicated that on a µg PM basis, flaming pine and eucalyptus were the most irritating. In contrast, on an emission-factor basis, which normalizes responses to the amount of PM produced/kg of fuel burned, smoldering smoke condensates induced greater irritant responses (>100-fold) than flaming smoke condensates, with smoldering pine being the most potent. Importantly, irritant responses significantly correlated with polycyclic aromatic hydrocarbon (PAH) content, but not with organic carbon or methoxyphenols. Data indicate that fuel type and burn condition influence the quantity and chemical composition of PM as well as toxicity.

Comparative electrocardiographic, autonomic and systemic inflammatory responses to soy biodiesel and petroleum diesel emissions in rats.

CONTEXT: Biodiesel fuel represents an alternative to high particulate matter (PM)-emitting petroleum-based diesel fuels, yet uncertainty remains regarding potential biodiesel combustion emission health impacts. OBJECTIVE: The purpose of this study was to compare cardiovascular responses to pure and blended biodiesel fuel emissions relative to petroleum diesel exhaust (DE). MATERIALS AND METHODS: Spontaneously hypertensive rats were exposed for 4 h per day for four days via whole body inhalation to combustion emissions (based on PM concentrations 50, 150 or 500 µg/m(3) or filtered air) from pure (B100) or blended (B20) soy biodiesel, or to pure petroleum DE (B0). Electrocardiogram (ECG) and heart rate variability (HRV, an index of autonomic balance) were monitored before, during and after exposure while pulmonary and systemic inflammation were assessed one day after the final exposure. ECG and HRV data and inflammatory data were statistically analyzed using a linear mixed model for repeated measures and an analysis of variance, respectively. RESULTS: B100 and B0, but not B20, increased HRV during all exposure days at the highest concentration indicating increased parasympathetic tone. Electrocardiographic data were mixed. B100 and B0, but not B20, caused significant changes in one or more of the following: serum C-reactive protein, total protein, low density lipoprotein (LDL) and high density lipoprotein (HDL) cholesterol, and blood urea nitrogen (BUN) and plasma angiotensin converting enzyme (ACE) and fibrinogen. DISCUSSION AND CONCLUSIONS: Although responses to emissions from all fuels were mixed and relatively mild, some findings point to a reduced cardiovascular impact of blended biodiesel fuel emissions.

Attenuated allergic responses to house dust mite antigen in feed-restricted rats.

Caloric restriction has been shown to alter a broad range of immunological end points in both experimental animals and humans. The objective of this study was to investigate the effect of short-term moderate feed restriction (25% reduction) on allergic immune responses in Brown Norway rats. After 3 weeks of acclimation to their feed regimens, rats were sensitized and 2 weeks later challenged with house dust mite (HDM) antigen via intratracheal instillation. Feed restriction resulted in lower levels of antigen-specific IgE in serum and reduced antigen specific lymphoproliferative activity in pulmonary lymph nodes. Feed restriction also attenuated pulmonary inflammation, as evidenced by lower levels of lactate dehydrogenase and total protein, decreased infiltration of neutrophils and eosinophils, and reduced secretion of pro-inflammatory cytokine tumor necrosis factor (TNF)-[alpha] in bronchoalveolar lavage fluid. In addition, feed restriction decreased TNF-[alpha] secretion in serum and decreased mRNA expression of TNF-[alpha] and interleukin-6 in pulmonary lymph nodes. We conclude that feed restriction strongly dampened the allergic immune responses to HDM in rats and that this attenuation was associated with decreased expression and secretion of pro-inflammatory cytokines.

The effects of ozone on immune function.

A review of the literature reveals that ozone (O3) exposure can either suppress or enhance immune responsiveness. These disparate effects elicited by O3 exposure depend, in large part, on the experimental design used, the immune parameters examined as well as the animal species studied. Despite the apparent contradictions, a general pattern of response to O3 exposure can be recognized. Most studies indicate that continuous O3 exposure leads to an early (days 0-3) impairment of immune responsiveness followed, with continued exposures, by a form of adaptation to O3 that results in a re-establishment of the immune response. The effects of O3 exposure on the response to antigenic stimulation also depend on the time at which O3 exposure occurred. Whereas O3 exposure prior to immunization is without effect on the response to antigen, O3 exposure subsequent to immunization suppresses the response to antigen. Although most studies have focused on immune responses in the lung, numerous investigators have provided functional and anatomical evidence to support the hypothesis that O3 exposure can have profound effects on systemic immunity.

Air pollutant-enhanced respiratory disease in experimental animals.

Studies in animals have shown that a wide range of airborne particulates including cigarette smoke, acid aerosols, metals, organic compounds, and combustion products can interfere with the normal defense processes of the lung to enhance susceptibility to respiratory infection or exacerbate allergic diseases. Such detrimental effects are less easy to quantify in humans because of the difficulties in obtaining comprehensive exposure history and health status in large populations and because of the inherent dangers of inducing disease in clinical studies. In this article we describe examples of how air pollutants affect lung disease in experimental animal systems. This information can be used to predict the health risk of simple and complex exposures and to lend insight into the mechanisms of air pollution toxicity.

Suppression of alveolar macrophage membrane-receptor-mediated phagocytosis by model particle-adsorbate complexes: physicochemical moderators of uptake.

In order to assess the abilities of alveolar macrophages (AMs) to phagocytize adsorbent-adsorbate complexes, rat AMs were incubated in vitro with two carbon blacks that have 15-fold differences in specific surface areas (ASTM classification N339 less than Black Pearls 2000) sorbed with 0.5 and 1.0 monolayer coverages of a polar and semi-polar adsorbate (acrolein and benzofuran, respectively). One-half monolayer coverages of N339 with either adsorbates significantly suppressed the phagocytosis of the carbon black, whereas one monolayer coverage did not. Neither adsorbate at either coverages affected the phagocytosis of Black Pearls 2000. The capacity of macrophages to phagocytize a subsequent particle challenge via the Fc-membrane receptor was quantified following treatment of the macrophages with the carbon black-adsorbate complexes. Treatment of the macrophages with carbon black N339-adsorbates complexes at both coverages impaired Fc-receptor-mediated phagocytosis, whereas no effect was observed when the carbon black was Black Pearls 2000. The results of this study indicate that the surface properties of the particles, the chemical properties of the chemical pollutants, and the interactions between particles and pollutants play a major role in defining the biological effect of particle-pollutant complexes.

Animal models to assess the effects of air pollutants on allergic lung disease.

Animal models provide toxicologists with useful tools for assessing risks associated with respiratory allergy. Both the mouse and BN rat models described exhibit many of the features of human allergic asthma. It is clear that environmental contaminants can exacerbate the expression of these features. Work is under way to explore underlying mechanisms and to develop methods for applying these data to human health risk assessment.

Increased cholinergic antagonism underlies impaired beta-adrenergic response in ovalbumin-sensitized guinea pigs.

The goal of this study was to determine if the hyporesponsiveness to beta-adrenoceptor stimulation observed in ovalbumin-sensitized tracheal smooth muscle is due to increased cholinergic muscarinic tone or to a defect in the beta-adrenergic cascade itself. We examined the effects of ovalbumin-sensitization on the responsiveness of guinea pig tracheae to agents that mediate relaxation at various steps in the beta-adrenergic cascade when the tracheal tissue was preconstricted with either carbachol or histamine. Ovalbumin sensitization caused significant reductions in the maximal relaxations both to the beta-adrenergic agonist isoproterenol and to prostaglandin E2 (PGE2) in guinea pig trachealis when the tracheal tissue was preconstricted with the muscarinic agonist carbachol. In contrast, sensitization had no effect on the ability of PGE2 and isoproterenol to relax histamine contractions. Preconstricting the tissues with increasing concentrations of KCl reduced the effectiveness of isoproterenol to relax equally airway tissues from both sensitized and control animals. Forskolin-induced relaxations of trachealis muscle were not altered with sensitization. When tracheal tissues were precontracted with increasing concentrations of carbachol, the effectiveness of isoproterenol and PGE2 to relax airway tissues decreased. Functional antagonism of relaxations by muscarinic agonists was enhanced in the sensitized tissues, since the concentration of carbachol necessary to reduce beta-adrenoceptor-induced relaxations to the same degree as in the control animals was a log dose lower. These results suggest that the impaired beta-adrenoceptor response in sensitized tissues is not due to an intrinsic defect in the beta-adrenergic cascade but to an enhancement of a muscarinic cholinergic pathway.

Enhanced allergic responses to house dust mite by oral exposure to carbaryl in rats.

Epidemiological studies have demonstrated an association between use of carbamate insecticides, including carbaryl, and increased incidence of allergic asthma in farmers. In this study the effect of oral carbaryl exposure on the development of allergic responses to house dust mite (HDM) was examined in female Brown Norway rats. Rats were gavaged for 2 weeks with 0, 2, 10, or 50 mg/kg/day of carbaryl. They were sensitized with a subcutaneous injection of HDM in aluminum hydroxide adjuvant 3 days after the beginning of carbaryl exposure and challenged with antigen via the trachea 1 day after the final carbaryl ingestion. Two days after challenge, antigen-specific cell proliferation in pulmonary lymph nodes was significantly higher in the 50 mg/kg group than in controls, while antigen-specific splenocyte proliferation was decreased in groups dosed with 2, 10, and 50 mg/kg carbaryl. Total protein and lymphocyte number in bronchoalveolar lavage (BAL) fluid were also increased in the 50 mg/kg group. By 7 days after challenge, immune-mediated pulmonary inflammation (eosinophils), antigen-specific immunoglobulin (Ig) E level in serum, and antigen-specific IgE and IgA levels in BAL fluid were significantly elevated in the 50 mg/kg group. No apparent change was observed for lactate dehydrogenase and eosinophil peroxidase in BAL fluid, while the number of BAL macrophages were decreased in groups dosed with 10 and 50 mg/kg carbaryl. The results suggest that carbaryl may cause systemic immune suppression, while enhancing pulmonary allergic responses to house dust mite antigen.

Suppression of allergic immune responses to house dust mite (HDM) in rats exposed to 2,3,7,8-TCDD.

Exposure to various xenobiotics, including oxidant gases, diesel exhaust, and certain pesticides, has been reported to exacerbate pulmonary allergic hypersensitivity responses. Increased lymphocyte proliferative responses to parasite antigens or increased antibody responses to sheep erythrocyte have also been reported in rats exposed to TCDD before infection or immunization. As a result, these studies were conducted to test the hypothesis that TCDD exposure exacerbates the allergic response to house dust mite antigen. Brown Norway rats were injected, ip, with 0, 1, 10, or 30 microg TCDD/kg 7 days before intratracheal (it) sensitization to semipurified house dust mite allergen (HDM). Fourteen days later, rats were challenged with HDM and immediate bronchospasm was measured. At this time point, plus 2 and 7 days later, inflammatory cells in bronchoalveolar lavage fluid (BALF), HDM-specific IgE levels in serum, and HDM-driven cell proliferation in bronchial lymph nodes and spleen were evaluated. TCDD exposure decreased both immediate bronchoconstriction and specific IgE synthesis after the HDM challenge; 7 days later, HDM-specific IgE responses remained suppressed. Total serum IgE levels were similar in all groups. HDM challenge alone significantly increased cellular and biochemical indicators of lung injury, both of which were suppressed by TCDD exposure. The proliferative response of lymph node cells, but not of spleen cells, to HDM was also suppressed at the highest TCDD dose, although the splenic response to Concanavalin A was elevated. It appears that early events in the response to HDM are affected by TCDD exposure, since message for IL5 was dramatically reduced 2 days after sensitization, but not after challenge. We therefore conclude that TCDD exposure suppressed, rather than enhanced the development of allergic immune responses and the expression of immune-mediated lung disease.

Exposure to ultraviolet radiation enhances mortality and pathology associated with influenza virus infection in mice.

Ultraviolet radiation (UVR) causes systemic immune suppression, decreasing the delayed type and contact hypersensitivity responses in animals and humans and enhancing certain mycobacterial, parasitic and viral infections in mice. This study tests the hypothesis that prior exposure to UVR enhances influenza infections in mice. BALB/c female mice were exposed to 0-8.2 kJ/m2 of UVR. Exposed and unexposed mice were infected intranasally three days later with 150-300 plaque-forming units/mouse (lethal dose (LD)20-LD40) of mouse-adapted Hong Kong Influenza A/68 (H3N2) virus or sham infected with 50 microL Hanks' balanced salt solution/mouse. Mortality from viral infection ranged from 25-50%. UVR exposure increased virus-associated mortality in a dose-dependent manner (up to a two-fold increase at 8.2 kJ/m2). The increased mortality was not associated with bacterial pneumonia. The highest dose of UVR also accelerated the body weight loss and increased the severity and incidence of thymic atrophy associated with influenza infection. However, UVR treatment had little effect on the increase in lung wet weight seen with viral infection, and, to our surprise, did not cause an increase in virus titers in the lung or dissemination of virus. The mice died 5-6 days after infection, too early for adaptive immune responses to have much impact. Also, UVR did not interfere with the development of protective immunity to influenza, as measured by reinfection with a lethal challenge of virus. Also, cells adoptively transferred from UVR or untreated mice were equally protective of recipient mice challenged with a lethal dose of virus. The mice resemble mice succumbing to endotoxin, and influenza infection increased the levels of tumor necrosis factor alpha (TNF-alpha) in bronchoalveolar lavage fluid and serum cortisol levels; however, UVR preexposure did not increase either of these responses to the virus. The results show that UVR increased the morbidity, mortality and pathogenesis of influenza virus in mice without affecting protective immunity to the virus, as measured by resistance to reinfection. The mechanism of enhanced mortality is uncertain, but the data raises concerns that UVR may exacerbate early responses that contribute to the pathogenesis of a primary viral infection.

Interaction of air pollutants and pulmonary allergic responses in experimental animals.

Asthma, which is primarily an allergic type of respiratory disease, has increased in the U.S. and Europe by 30% over the last decade. Air pollution may play a role in this rise, since during episodes of smog, hospital admissions due to asthma increase. Ambient air quality has generally improved since the Clean Air Act was implemented in 1971 however, and has led some investigators to suggest that the increased risk of asthma is associated with a deterioration of indoor air quality through the introduction of closed ventilation systems and constant climate control. Thus, although the direct health effects of acute and chronic air pollutant exposure are not in dispute, emphasis on the sources and location of exposure is changing from outdoors to the home environment and workplace. The few experimental studies which have investigated the interaction of air pollutants with allergic disease have shown that exposure to O3 or NO2 can increase levels of allergen-specific antibody and may augment allergic symptoms. These experiments are reviewed along with a study conducted in our laboratory which demonstrated the enhancing effect of NO2 exposure on immune responses and pulmonary inflammation following sensitization and pulmonary challenge with house dust mite allergen (HDM). In this study, rats exposed to 5 ppm NO2 for 3 h after each immunization had significantly higher levels of serum IgE and local IgA, IgG and IgE antibody than air controls. Lymphocyte activity in the spleen and local lymph nodes, and pulmonary inflammatory cells were also increased in NO2-exposed rats. The results show that exposure to NO2 enhances immune responsiveness and the severity of pulmonary inflammation following antigen challenge. Since allergic individuals and most asthmatics also have increased immunity to these proteins, the possibility that air pollutant exposure enhances immune responses to allergens and thus exacerbates immune-mediated lung disease exists.

Morphine reduces pulmonary inflammation in response to influenza infection.

The present study shows that morphine reduces the pulmonary inflammatory response to intranasal influenza virus infection in rats. Rats were infected with rat-adapted influenza virus (RAIV), which is a unique infectious agent because normal rats develop an acute pulmonary inflammatory response to RAIV and rapidly clear the virus within a few days with no mortality. Male Lewis rats were implanted with 75 mg morphine pellets or placebo pellets 72 hours prior to intranasal RAIV infection. Rats were euthanized at 2, 24, 48, 72, and 96 hours after infection. Assessment of inflammation included accumulation of inflammatory cells in the lungs, lung weight, and protein and LDH content of bronchial alveolar lavage fluid (BALF). Placebo-treated rats showed a marked inflammatory response to RAIV infection, and morphine-treated rats mounted less vigorous inflammatory responses to the infection. Taken together, these data suggest that morphine treatment impairs the inflammatory response to RAIV infection in the lungs, which is consistent with prior work demonstrating that morphine is a potent anti-inflammatory agent in other areas of the body.

The airborne survival of Pasteurella haemolytica and its deposition in and clearance from the mouse lung.

Pasteurella haemolytica A1 was aerosolised by a Collison nebuliser in a Henderson apparatus and its survival in air was measured. The organism was fragile in aerosol and survived best at high humidity and warm temperature. Mice were exposed to the aerosol and clearance from the lung measured. Deposition in the mouse lung showed a good linear correlation with bacterial concentration in the spray suspension fluid. Clearance from the lung was rapid over 24 h although some bacteria could be detected 2 and 4 days after exposure. Mice which received a second exposure 2 weeks later exhibited accelerated clearance from the lung whereby no bacteria could be detected after 12 h. This was associated with serum IgG antibody production, and local and splenic lymphocyte responses to bacterial antigen in vitro.

Morphine alters the immune response to influenza virus infection in Lewis rats.

Although the in vitro immunomodulatory effects of morphine are well-documented, few studies have explored the impact of morphine on viral infection in intact rats. We report that morphine can alter in vivo immune responsiveness to pulmonary influenza virus infection in Lewis rats. We studied rat-adapted influenza virus (RAIV) infection, which is a unique infectious disease system because normal rats develop an acute inflammatory response to RAIV in the lung, and rapidly clear the virus within a few days, with no mortality (13,20,21). Male Lewis rats were implanted with 75 mg morphine pellets or placebo pellets 72 hours prior to intranasal RAIV infection. Rats were euthanized at 2, 24, 48, 72 and 96 hours after infection and inflammation and viral load were measured in the lungs. Placebo-treated rats showed marked inflammatory responses to RAIV infection, and quickly cleared the virus from their lungs. Morphine-treated rats mounted less vigorous inflammatory responses to the infection and cleared the virus more slowly than placebo-treated rats. Although these initial data indicate that morphine can alter the response to RAIV, additional studies are necessary to fully characterize these effects.

A comparison of the pulmonary defenses against streptococcal infection in rats and mice following O3 exposure: differences in disease susceptibility and neutrophil recruitment.

Ozone (O3) exposure reduces alveolar macrophage (AM) phagocytosis in mice and increases their susceptibility to Streptococcus zooepidemicus. O3 exposure also decreases AM phagocytosis in rats but does not result in mortality to infection. To investigate the mechanism of disease protection in rats, antibacterial defenses of two strains of mice and F344 rats were compared. O3 exposure (3 hr, 0.4 or 0.8 ppm) and infection with S. zooepidemicus resulted in a dose-dependent proliferation of bacteria in the lungs of mice and high mortality. Polymorphonuclear leukocytes (PMNs) were observed in severely affected individuals 2 or more days postinfection and did not alter the fatal infection. In contrast, microbial inactivation was only impaired in O3-exposed rat lungs during the first 48 hr after infection. In these animals PMNs could be isolated from bronchoalveolar lavage fluid between 6 and 48 hr postinfection with the peak response occurring at 24 hr. Pretreatment with anti-PMN serum eliminated the neutrophil influx and impaired further the bactericidal activity in ozone-exposed rats. The results suggest that inhaled streptococci are cleared normally from the mouse lung by AMs. Following exposure to O3, AM phagocytosis is reduced and the mice develop a fatal infection. The persistence of bacteria in the lungs of O3-exposed rats triggers a transient influx of PMNs whose appearance corresponds with elimination of the bacteria. Differences in antimicrobial defenses between various experimental species and humans need to be better understood in order to predict effects of air pollutants on susceptibility to infection in man.

Enhanced Allergic Sensitization in Animals Exposed to Particulate Air Pollution.

Epidemiological studies have found an association between elevated levels of particulate matter (PM) air pollution and increased medication use and hospital visits by asthmatics. While it is known that asthmatics are generally more sensitive to airborne contaminants such as sulfur dioxide and tobacco smoke, it is difficult to test which components of air pollution may also contribute to the induction of pulmonary allergy (sensitization) because of the risk in creating disease. Recent studies in mice and rats, however, have demonstrated that pulmonary exposure to combustion particles such as diesel and residual oil fly ash (ROFA) can exacerbate immunological sensitization (in the form of immunoglobulin E antibody and lymphocyte reactivity) to experimental and natural allergens. Subsequent allergen challenge in these animals results in a greater allergen-induced bronchoconstriction, elevated numbers of eosinophils in the lung, and enhanced airway responsiveness to cholinergic agents compared to what occurs in similarly immunized animals pretreated with vehicle or "inert" particles. Although the mechanisms for these effects are not known, it has been demonstrated that the adjuvant effects of diesel and ROFA can be reproduced with hydrocarbons and soluble transition metals from diesel and ROFA, respectively. In addition, analysis of mediator expression and release over the sensitization phase has revealed that PM exposure can enhance production of Th2 cytokines such as interleukin-5 (IL-5) and the proinflammatory cytokine tumor necrosis factor-alpha (TNF-α). These experimental systems demonstrate the potential of particulate air pollutants to enhance allergic sensitization and can be further used to elucidate the mechanism for these effects.

Asthma and PM10.

PM10 (the mass of particles present in the air having a 50% cutoff for particles with an aerodynamic diameter of 10 microm) is the standard measure of particulate air pollution used worldwide. Epidemiological studies suggest that asthma symptoms can be worsened by increases in the levels of PM10. Epidemiological evidence at present indicates that PM10 increases do not raise the chances of initial sensitisation and induction of disease, although further research is warranted. PM10 is a complex mixture of particle types and has many components and there is no general agreement regarding which component(s) could lead to exacerbations of asthma. However pro-inflammatory effects of transition metals, hydrocarbons, ultrafine particles and endotoxin, all present to varying degrees in PM10, could be important. An understanding of the role of the different components of PM10 in exacerbating asthma is essential before proper risk assessment can be undertaken leading to advice on risk management for the many asthmatics who are exposed to air pollution particles.

Ozone-enhanced pulmonary infection with Streptococcus zooepidemicus in mice. The role of alveolar macrophage function and capsular virulence factors.

Ozone exposure has been shown to increase the susceptibility of mice to pulmonary bacterial infection. We report here the differences in susceptibility of two strains of mice (C3H/HeJ and C57Bl/6) to pulmonary challenge with Streptococcus zooepidemicus, and demonstrate an association between O3 exposure, reduced alveolar macrophage (AM) function, and increased mortality to infection. After a 3-h exposure to air or to 0.4 or 0.8 ppm O3, mice received an infection of bacteria by aerosol. Subsequent mortality observed over a 20-day period for any given exposure concentration was greater in the C3H/HeJ mice than in the C57Bl/6 mice. Phagocytosis assays identified the AM from O3-exposed lungs as having an impaired ability to engulf the bacteria. Baseline phagocytic activity in C3H/HeJ mice was lower than that in C57Bl/6 mice. Microbiologic assessment of the lungs at various times after infection revealed that the streptococci proliferated rapidly in the lungs of O3-exposed mice, grew more quickly upon isolation, and displayed a mucoid colony appearance indicative of increased encapsulation. In vitro assays confirmed that the encapsulated isolates prevented binding of the bacteria to AM, and reinfection of nonexposed mice with the encapsulated isolate resulted in increased mortality compared with infection with similar numbers of the original unencapsulated bacteria. We have demonstrated that O3 inhalation impairs AM activity in the lung. The streptococci are then able to proliferate and more fully express virulence factors, in particular, the antiphagocytic capsule, which prohibits the ingestion of bacteria by pulmonary phagocytes and leads to increased severity of infection.