Systemic immunological responses are dependent on sex and ovarian hormone presence following acute inhaled woodsmoke exposure.

BACKGROUND: Rural regions of the western United States have experienced a noticeable surge in both the frequency and severity of acute wildfire events, which brings significant challenges to both public safety and environmental conservation efforts, with impacts felt globally. Identifying factors contributing to immune dysfunction, including endocrinological phenotypes, is essential to understanding how hormones may influence toxicological susceptibility. METHODS: This exploratory study utilized male and female C57BL/6 mice as in vivo models to investigate distinct responses to acute woodsmoke (WS) exposure with a focus on sex-based differences. In a second set of investigations, two groups were established within the female mouse cohort. In one group, mice experienced ovariectomy (OVX) to simulate an ovarian hormone-deficient state similar to surgical menopause, while the other group received Sham surgery as controls, to investigate the mechanistic role of ovarian hormone presence in driving immune dysregulation following acute WS exposure. Each experimental cohort followed a consecutive 2-day protocol with daily 4-h exposure intervals under two conditions: control HEPA-filtered air (FA) and acute WS to simulate an acute wildfire episode. RESULTS: Metals analysis of WS particulate matter (PM) revealed significantly increased levels of 63Cu, 182W, 208Pb, and 238U, compared to filtered air (FA) controls, providing insights into the specific metal components most impacted by the changing dynamics of wildfire occurrences in the region. Male and female mice exhibited diverse patterns in lung mRNA cytokine expression following WS exposure, with males showing downregulation and females displaying upregulation, notably for IL-1ß, TNF-α, CXCL-1, CCL-5, TGF-ß, and IL-6. After acute WS exposure, there were notable differences in the responses of macrophages, neutrophils, and bronchoalveolar lavage (BAL) cytokines IL-10, IL-6, IL-1ß, and TNF-α. Significant diverse alterations were observed in BAL cytokines, specifically IL-1ß, IL-10, IL-6, and TNF-α, as well as in the populations of immune cells, such as macrophages and polymorphonuclear leukocytes, in both Sham and OVX mice, following acute WS exposure. These findings elucidated the profound influence of hormonal changes on inflammatory outcomes, delineating substantial sex-related differences in immune activation and revealing altered immune responses in OVX mice due to ovarian hormone deficiency. In addition, the flow cytometry analysis highlighted the complex interaction between OVX surgery, acute WS exposure, and their collective impact on immune cell populations within the hematopoietic bone marrow niche. CONCLUSIONS: In summary, both male and female mice, alongside females subjected to OVX and those who had sham surgery, exhibit significant variations in the expression of proinflammatory cytokines, chemokines, lung mRNA gene expression, and related functional networks linked to signaling pathways. These differences potentially act as mediators of sex-specific and hormonal influences in the systemic inflammatory response to acute WS exposure during a wildfire event. Understanding the regulatory roles of genes expressed differentially under environmental stressors holds considerable implications, aiding in identifying sex-specific therapeutic targets for addressing acute lung inflammation and injury.

Acute inhalation of tungsten particles results in early signs of cardiac injury.

Epidemiological studies have established that exposure to tungsten increases the risk of developing cardiovascular diseases. However, no studies have investigated how tungsten affects cardiac function or the development of cardiovascular disease. Inhalation of tungsten particulates is relevant in occupational settings, and inhalation of particulate matter has a known causative role in driving cardiovascular disease. This study examined if acute inhalation to tungsten particulates affects cardiac function and leads to heart tissue alterations. Female BALB/c mice were exposed to Filtered Air or 1.5 ± 0.23 mg/m3 tungsten particles, using a whole-body inhalation chamber, 4 times over the course of two weeks. Inhalation exposure resulted in mild pulmonary inflammation characterized by an increased percentage and number of macrophages and metabolomic changes in the lungs. Cardiac output was significantly decreased in the tungsten-exposed group. Additionally, A', an indicator of the amount of work required by the atria to fill the heart was elevated. Cardiac gene expression analysis revealed, tungsten exposure increased expression of pro-inflammatory cytokines, markers of remodeling and fibrosis, and oxidative stress genes. These data strongly suggest exposure to tungsten results in cardiac injury characterized by early signs of diastolic dysfunction. Functional findings are in parallel, demonstrating cardiac oxidative stress, inflammation, and early fibrotic changes. Tungsten accumulation data would suggest these cardiac changes are driven by systemic consequences of pulmonary damage.

Inhalation of Tungsten Metal Particulates Alters the Lung and Bone Microenvironments Following Acute Exposure.

Inhalation of tungsten particulates is a relevant route of exposure in occupational and military settings. Exposure to tungsten alloys is associated with increased incidence of lung pathologies, including interstitial lung disease and cancer. We have demonstrated, oral exposure to soluble tungsten enhances breast cancer metastasis to the lungs through changes in the surrounding microenvironment. However, more research is required to investigate if changes in the lung microenvironment, following tungsten particulate exposure, can drive tumorigenesis or metastasis to the lung niche. This study examined if inhalation to environmentally relevant concentrations of tungsten particulates caused acute damage to the microenvironment in the lungs and/or systemically using a whole-body inhalation system. Twenty-four female BALB/c mice were exposed to Filtered Air, 0.60 mg/m3, or 1.7 mg/m3 tungsten particulates (<1 µm) for 4 h. Tissue samples were collected at days 1 and 7 post-exposure. Tungsten accumulation in the lungs persisted up to 7 days post-exposure and produced acute changes to the lung microenvironment including increased macrophage and neutrophil infiltration, increased levels of proinflammatory cytokines interleukin 1 beta and C-X-C motif chemokine ligand 1, and an increased percentage of activated fibroblasts (alpha-smooth muscle actin+). Exposure to tungsten also resulted in systemic effects on the bone, including tungsten deposition and transient increases in gene expression of proinflammatory cytokines. Taken together, acute whole-body inhalation of tungsten particulates, at levels commonly observed in occupational and military settings, resulted in changes to the lung and bone microenvironments that may promote tumorigenesis or metastasis and be important molecular drivers of other tungsten-associated lung pathologies such as interstitial lung disease.

Revised fish aging techniques improve fish contaminant trend analyses in the face of changing Great Lakes food webs.

Incorporation of fish age into the assessment of status and trends for persistent, bioaccumulative and toxic chemicals in the Great Lakes has become an important step for the U.S. EPA's Great Lakes Fish Monitoring and Surveillance Program (GLFMSP). A slowing in the rate of decline for total PCBs in Lake Huron beginning in 2000, led the Program to complete a retrospective analysis to assess how chemical contamination may be influenced by fish age. Analytical results suggest that fish age is an important variable when assessing contaminant trends and that the Program needed to revise its compositing scheme to group fish according to age, rather than by length, prior to homogenization and chemical analysis. An Interlaboratory comparison study of multiple age structures was performed to identify the most appropriate age estimation structure for the Program. The lake trout (Salvelinus namaycush) maxillae was selected, over the otolith, as the most precise, accurate, and rapidly assessed structure for the Program when compared between laboratories and against the known age from the coded wire tag (CWT). Age-normalization practices can now be implemented when assessing contaminant concentrations and trends for the GLFMSP.

Chronic carcinogenicity study of gasoline vapor condensate (GVC) and GVC containing methyl tertiary-butyl ether in F344 rats.

Chronic inhalation studies were conducted to compare the toxicity and potential carcinogenicity of evaporative emissions from unleaded gasoline (GVC) and gasoline containing the oxygenate methyl tertiary-butyl ether (MTBE; GMVC). The test materials were manufactured to mimic vapors people would be exposed to during refueling at gas stations. Fifty F344 rats per gender per exposure level per test article were exposed 6 h/d, 5 d/wk for 104 wk in whole body chambers. Target total vapor concentrations were 0, 2, 10, or 20 g/m³ for the control, low-, mid-, and high-level exposures, respectively. Endpoints included survival, body weights, clinical observations, organs weights, and histopathology. GVC and GMVC exerted no marked effects on survival or clinical observations and few effects on organ weights. Terminal body weights were reduced in all mid- and high-level GVC groups and high-level GMVC groups. The major proliferative lesions attributable to gasoline exposure with or without MTBE were renal tubule adenomas and carcinomas in male rats. GMV exposure led to elevated testicular mesothelioma incidence and an increased trend for thyroid carcinomas in males. GVMC inhalation caused an increased trend for testicular tumors with exposure concentration. Mid- and high-level exposures of GVC and GMVC led to elevated incidences of nasal respiratory epithelial degeneration. Overall, in these chronic studies conducted under identical conditions, the health effects in F344 rats following 2 yr of GVC or GMVC exposure were comparable in the production of renal adenomas and carcinomas in male rats and similar in other endpoints.

Levofloxacin cures experimental pneumonic plague in African green monkeys.

BACKGROUND: Yersinia pestis, the agent of plague, is considered a potential bioweapon due to rapid lethality when delivered as an aerosol. Levofloxacin was tested for primary pneumonic plague treatment in a nonhuman primate model mimicking human disease. METHODS AND RESULTS: Twenty-four African Green monkeys (AGMs, Chlorocebus aethiops) were challenged via head-only aerosol inhalation with 3-145 (mean = 65) 50% lethal (LD(50)) doses of Y. pestis strain CO92. Telemetered body temperature >39 °C initiated intravenous infusions to seven 5% dextrose controls or 17 levofloxacin treated animals. Levofloxacin was administered as a "humanized" dose regimen of alternating 8 mg/kg and 2 mg/kg 30-min infusions every 24-h, continuing until animal death or 20 total infusions, followed by 14 days of observation. Fever appeared at 53-165 h and radiographs found multilobar pneumonia in all exposed animals. All control animals died of severe pneumonic plague within five days of aerosol exposure. All 16 animals infused with levofloxacin for 10 days survived. Levofloxacin treatment abolished bacteremia within 24 h in animals with confirmed pre-infusion bacteremia, and reduced tachypnea and leukocytosis but not fever during the first 2 days of infusions. CONCLUSION: Levofloxacin cures established pneumonic plague when treatment is initiated after the onset of fever in the lethal aerosol-challenged AGM nonhuman primate model, and can be considered for treatment of other forms of plague. Levofloxacin may also be considered for primary presumptive-use, multi-agent antibiotic in bioterrorism events prior to identification of the pathogen.

Primary pneumonic plague in the African Green monkey as a model for treatment efficacy evaluation.

BACKGROUND: Primary pneumonic plague is rare among humans, but treatment efficacy may be tested in appropriate animal models under the FDA 'Animal Rule'. METHODS: Ten African Green monkeys (AGMs) inhaled 44-255 LD(50) doses of aerosolized Yersinia pestis strain CO92. Continuous telemetry, arterial blood gases, chest radiography, blood culture, and clinical pathology monitored disease progression. RESULTS: Onset of fever, >39°C detected by continuous telemetry, 52-80 hours post-exposure was the first sign of systemic disease and provides a distinct signal for treatment initiation. Secondary endpoints of disease severity include tachypnea measured by telemetry, bacteremia, extent of pneumonia imaged by chest x-ray, and serum lactate dehydrogenase enzyme levels. CONCLUSIONS: Inhaled Y. pestis in the AGM results in a rapidly progressive and uniformly fatal disease with fever and multifocal pneumonia, serving as a rigorous test model for antibiotic efficacy studies.

Milestones in progression of primary pneumonic plague in cynomolgus macaques.

Vaccines against primary pneumonic plague, a potential bioweapon, must be tested for efficacy in well-characterized nonhuman primate models. Telemetered cynomolgus macaques (Macaca fascicularis) were challenged by the aerosol route with doses equivalent to approximately 100 50% effective doses of Yersinia pestis strain CO92 and necropsied at 24-h intervals postexposure (p.e.). Data for telemetered heart rates, respiratory rates, and increases in the temperature greater than the diurnal baseline values identified the onset of the systemic response at 55 to 60 h p.e. in all animals observed for at least 70 h p.e. Bacteremia was detected at 72 h p.e. by a Yersinia 16S rRNA-specific quantitative reverse transcription-PCR and was detected later by the culture method at the time of moribund necropsy. By 72 h p.e. multilobar pneumonia with diffuse septal inflammation consistent with early bacteremia was established, and all lung tissues had a high bacterial burden. The levels of cytokines or chemokines in serum were not significantly elevated at any time, and only the interleukin-1beta, CCL2, and CCL3 levels were elevated in lung tissue. Inhalational plague in the cynomolgus macaque inoculated by the aerosol route produces most clinical features of the human disease, and in addition the disease progression mimics the disease progression from the anti-inflammatory phase to the proinflammatory phase described for the murine model. Defined milestones of disease progression, particularly the onset of fever, tachypnea, and bacteremia, should be useful for evaluating the efficacy of candidate vaccines.

Carcinogenic interactions between a single inhalation of 239PuO2 and chronic exposure to cigarette smoke in rats.

Rats were exposed once by inhalation to plutonium-239 dioxide ((239)PuO(2)), resulting in chronic alpha-particle irradiation of the lung, and exposed chronically to cigarette smoke to examine carcinogenic interactions between the two exposures. F344 rats were exposed to (239)PuO(2) to achieve an initial lung burden of 0.5 kBq and then exposed 6 h/day, 5 days/week to cigarette smoke at 100 or 250 mg particulate matter/m(3) for up to 30 months. Exposure to cigarette smoke increased the cumulative radiation dose to lung by slowing the clearance of (239)PuO(2). (239)PuO(2) alone did not affect survival, but the higher cigarette smoke exposure shortened survival in females. Combined exposure to (239)PuO(2) and cigarette smoke acted synergistically to shorten survival in both genders. The combined effects of cigarette smoke and (239)PuO(2) were approximately additive for lung hyperplasia and adenomas but were strongly synergistic for carcinomas. Differences between observed incidences and incidences predicted by survival-adjusted models accounting for increased radiation dose revealed a substantial component of synergy for carcinomas above that attributable to the radiation dose effect. The synergy for malignant lung tumors is consistent with findings from uranium miners and nuclear weapons production workers. These results bolster confidence in the epidemiological findings and have implications for risk assessment.

Generation and characterization of gasoline engine exhaust inhalation exposure atmospheres.

Exposure atmospheres for a rodent inhalation toxicology study were generated from the exhaust of a 4.3-L gasoline engine coupled to a dynamometer and operated on an adapted California Unified Driving Cycle. Exposure levels were maintained at three different dilution rates. One chamber at the lowest dilution had particles removed by filtration. Each exposure atmosphere was characterized for particle mass, particle number, particle size distribution, and detailed chemical speciation. The majority of the mass in the exposure atmospheres was gaseous carbon monoxide, nitrogen oxides, and volatile organics, with small amounts of particle-bound carbon/ions and metals. The atmospheres varied according to the cycle, with the largest spikes in volatile organic and inorganic species shown during the "cold start" portion of the cycle. Ammonia present from the exhaust and rodents interacted with the gasoline exhaust to form secondary inorganic particles, and an increase in exhaust resulted in higher proportions of secondary inorganics as a portion of the total particle mass. Particle size had a median of 10-20 nm by number and approximately 150 nm by mass. Volatile organics matched the composition of the fuel, with large proportions of aliphatic and aromatic hydrocarbons coupled to low amounts of oxygenated organics. A new measurement technique revealed organics reacting with nitrogen oxides have likely resulted in measurement bias in previous studies of combustion emissions. Identified and measured particle organic species accounted for about 10% of total organic particle mass and were mostly aliphatic acids and polycyclic aromatic hydrocarbons.

Development of a head-out plethysmograph system for non-human primates in an Animal Biosafety Level 3 facility.

INTRODUCTION: The term 'select agent' (SA) refers to a list of microorganisms and toxins and are defined as those that have the potential to pose a severe threat to public health and safety (42 C.F.R. Part 73). In order to carry out a research with SAs, an Animal Biosafety Level 3 (ABSL3) containment facility is required. Our newly completed ABSL3 facility is developing protocols for implementing safety and efficacy studies of therapeutics for SAs. METHODS: The primary purpose of this study was to develop methods for exposing non-human primates (NHP) to aerosolized SAs in the ABSL3 and systematically measure specific ventilatory endpoints (frequency, tidal volume, minute volume, and accumulated volume) using a head-out plethysmograph to more precisely control dosimetry. This report details the equipment and protocols used to conduct such studies within a containment facility. RESULTS: After validating the performance of the plethysmography system, we successfully exposed NHPs to an agent using the integrated plethysmography system. The system enabled an acquisition and analysis of ventilatory characteristics, facilitating accurate estimations of the inhaled dose. DISCUSSION: This system will have clear uses in the development of novel therapeutics and vaccines for the treatment of SAs in NHPs.

Particle size and composition related to adverse health effects in aged, sensitive rats.

Small increases in concentrations of ambient particulate matter (PM*) have been linked to adverse health effects, especially in older people and people with preexisting respiratory disease. Some epidemiologic studies have shown the association to be stronger with PM less than 2.5 microm in aerodynamic diameter (PM2.5) than with PM less than 10 microm in aerodynamic diameter (PM10). Some scientists and regulators suggest that 2.5 microm might be an arbitrary cutoff and that the effects might be more pronounced for PM less than 0.1 microm in aerodynamic diameter (ultrafine PM). Our first aim was to determine the relation between size of respirable particles and particle toxicity, as well as the health effects of short-term increases (spikes) in particle concentration against backgrounds of relatively low or high baseline exposures. Our second aim was to determine the effect of spikes in concentration of fine particles (0.7 microm in mass median aerodynamic diameter [MMAD]) and ultrafine particles (35 nm in count median diameter [CMD]) of disparate composition: vanadium pentoxide (V2O5) and carbon black. The relative toxicity of these particles was determined in aged rats with mild pulmonary inflammation induced by instilled endotoxin. Our third aim was to determine the influence of age (aged vs young adult) on particle-induced toxicity in these rats.

Life-span inhalation exposure to mainstream cigarette smoke induces lung cancer in B6C3F1 mice through genetic and epigenetic pathways.

Although cigarette smoke has been epidemiologically associated with lung cancer in humans for many years, animal models of cigarette smoke-induced lung cancer have been lacking. This study demonstrated that life time whole body exposures of female B6C3F1 mice to mainstream cigarette smoke at 250 mg total particulate matter/m(3) for 6 h per day, 5 days a week induces marked increases in the incidence of focal alveolar hyperplasias, pulmonary adenomas, papillomas and adenocarcinomas. Cigarette smoke-exposed mice (n = 330) had a 10-fold increase in the incidence of hyperplastic lesions, and a 4.6-fold (adenomas and papillomas), 7.25-fold (adenocarcinomas) and 5-fold (metastatic pulmonary adenocarcinomas) increase in primary lung neoplasms compared with sham-exposed mice (n = 326). Activating point mutations in codon 12 of the K-ras gene were identified at a similar rate in tumors from sham-exposed mice (47%) and cigarette smoke-exposed mice (60%). The percentages of transversion and transition mutations were similar in both the groups. Hypermethylation of the death associated protein (DAP)-kinase and retinoic acid receptor (RAR)-beta gene promoters was detected in tumors from both sham- and cigarette smoke-exposed mice, with a tendency towards increased frequency of RAR-beta methylation in the tumors from the cigarette smoke-exposed mice. These results emphasize the importance of the activation of K-ras and silencing of DAP-kinase and RAR-beta in lung cancer development, and confirm the relevance of this mouse model for studying lung tumorigenesis.

The toxicity of microcystin LR in mice following 7 days of inhalation exposure.

Microcystins, a family of cyclic heptapeptides produced by the cyanobacteria, Microcystis aeruginosa, have documented hepatotoxic and tumor promoting activities. The purpose of this study was to evaluate the toxicity of inhaled microcystin LR (microcystin). Male BALB/c mice were exposed by nose-only inhalation to 260-265 microg microcystin/m(3) for 7 days. The low-, mid- and high-dose groups were exposed for 0.5, 1, and 2h, respectively. Control animals were sham exposed to aerosolized vehicle. Treatment-related microscopic lesions were observed only in the nasal cavity of the mid- and high-dose groups. These lesions consisted of minimal to moderate multifocal degeneration and necrosis of the respiratory epithelium, with variable neutrophilic inflammation and minimal to marked degeneration, necrosis, and atrophy of the olfactory epithelium. The no-adverse-effect dose for the nasal lesions was approximately 3 microg/kg body weight, or 20 ng/cm(2) of nasal epithelium. In serum, only two protein peaks, occurring at m/zs of 11,688 and 11,829 Da, exhibited decreases in intensity that were microcystin dose-dependent. While these proteins have not been positively identified, they may be useful in the future as biomarkers of microcystin exposure in humans.

Effects of concentrated ambient particles on normal and hypersecretory airways in rats.

Epidemiological studies have reported that elevated levels of particulate air pollution in urban communities are associated with increases in attacks of asthma based on evidence from hospital admissions and emergency department visits. Principal pathologic features of chronic airway diseases, like asthma, are airway inflammation and mucous hypersecretion with excessive amounts of luminal mucus and increased numbers of mucus-secreting cells in regions of the respiratory tract that normally have few or no mucous cells (ie, mucous cell metaplasia). The overall goal of the present project was to understand the adverse effects of urban air fine particulate matter (PM2.5; < or = 2.5 pm in aerodynamic diameter)* on normal airways and airways compromised with airway inflammation and excess mucus. Our project was specifically designed to (1) examine the chemical and physical characteristics of PM2.5 and other airborne pollutants in the outdoor air of a local Detroit community with a high incidence of childhood asthma; (2) determine the effects of this community-based PM2.5 on the airway epithelium in normal rats and rats compromised with preexisting hypersecretory airway diseases (ie, animal models of human allergic airway disease--asthma and chronic bronchitis); and (3) identify the chemical or physical components of PM2.5 that are responsible for PM2.5 -induced airway inflammation and epithelial alterations in these animal models. Two animal models of airway disease were used to examine the effects of PM2.5 exposure on preexisting hypersecretory airways: neutrophilic airway inflammation induced by endotoxin challenge in F344 rats and eosinophilic airway inflammation induced by ovalbumin (OVA) challenge in BN rats. A mobile air monitoring and exposure laboratory equipped with inhalation exposure chambers for animal toxicology studies, air pollution monitors, and particulate collection devices was used in this investigation. The mobile laboratory was parked in a community in southwestern Detroit during the summer months when particulate air pollution is usually high (July and September 2000). We monitored the outdoor air pollution in this community daily, and exposed normal and compromised rats to concentrated PM2.5 from this local urban atmosphere. Rats in the inhalation studies were exposed for 1 day or for 4 or 5 consecutive days (10 hours/day) to either filtered air (controls) or concentrated ambient particles (CAPs) delivered by a Harvard ambient fine particle concentrator. Rats were killed 24 hours after the end of the exposure. Biochemical, morphometric, and molecular techniques were used to identify airway epithelial and inflammatory responses to CAPs. Lung lobes were also either intratracheally lavaged with saline to determine cellular composition and protein in bronchoalveolar lavage fluid (BALF) or removed for analysis by inductively coupled plasma-mass spectrometry (ICPMS) to detect retention of ambient PM2.5--derived trace elements. The Harvard concentrator effectively concentrated the fine ambient particles from this urban atmosphere (10-30 times) without significantly changing the major physicochemical features of the atmospheric particles. Daily CAPs mass concentrations during the 10-hour exposure period (0800-1800) in July ranged from 16 to 895 microg/m3 and in September ranged from 81 to 755 microg/m3. In general, chemical characteristics of ambient particles were conserved through the concentrator into the exposure chamber. Single or repeated exposures to CAPs did not cause adverse effects in the nasal or pulmonary airways of healthy F344 or BN rats. In addition, CAPs-related toxicity was not observed in F344 rats pretreated with bacterial endotoxin. Variable airway responses to CAPs exposure were observed in BN rats with preexisting allergic airway disease induced by OVA sensitization and challenge. Only OVA-challenged BN rats exposed to CAPs for 5 consecutive days in September 2000 had significant increases in airway mucosubstances and pulmonary inflammation compared to saline-challenged/air-exposed control rats. OVA-challenged BN rats that were repeatedly exposed to CAPs in July 2000 had only minor CAPs-related effects. In only the September 5-day exposure protocol, PM2.5 trace elements of anthropogenic origin (La, V, and S) were recovered from the lung tissues of CAPs-exposed rats. Recovery of these specific trace elements was greatest in rats with OVA-induced allergic airway disease. Additional laboratory experiments using intratracheal instillations of ambient PM2.5 samples were performed to identify bioactive agents in the CAPs to which rats had been exposed in the inhalation exposure component. Because the most pronounced effects of CAPs inhalation were found in BN rats with OVA-induced allergic airways exposed in September, we used ambient PM2.5 samples that were collected on 2 days during the September CAPs inhalation exposures to use for instillation. Ambient PM2.5 samples were collected, fractionated into soluble and insoluble species, and then compared with each other and with total PM2.5 for their effects in healthy BN rats and those with OVA-induced allergic airway disease. Intratracheal instillation of the insoluble fraction of PM2.5 caused mild neutrophilic inflammation in the lungs of healthy rats. However, total PM2.5 or the soluble or insoluble fractions instilled in rats with OVA-induced airway inflammation did not enhance the inflammation or the airway epithelial remodeling that was evident in some of the BN rats exposed to CAPs by inhalation. Therefore, the results from this instillation component did not suggest what fractions of the CAPs may have been responsible for enhancing OVA-induced airway mucosubstances and pulmonary inflammation observed in the inhalation exposure component. In summary, inhaled CAPs-related pulmonary alterations in the affected OVA-challenged rats appeared to be related to the chemical composition, rather than the mass concentration, to which the animals were exposed. Results of the trace element analysis in the lungs of CAPs-exposed BN rats exposed in September suggested that air particles derived from identified local combustion sources were preferentially retained in allergic airways. These results demonstrate that short-term exposures to CAPs from this southwestern Detroit community caused variable responses in laboratory rats and suggest that adverse biological responses to ambient PM2.5 may be associated more closely with local sources of particles and weather patterns than with particle mass.

Generation and characterization of four dilutions of diesel engine exhaust for a subchronic inhalation study.

Exposure atmospheres for a rodent inhalation toxicology study were generated from the exhaust of a 2000 Cummins ISB 5.9L diesel engine coupled to a dynamometer and operated on a slightly modified heavy-duty Federal Test Procedure cycle. Exposures were conducted to one clean air control and four diesel exhaust levels maintained at four different dilution rates (300:1, 100:1, 30:1, 10:1) that yielded particulate mass concentrations of 30, 100, 300, and 1000 microg/m3. Exposures at the four dilutions were characterized for particle mass, particle size distribution (reported elsewhere), detailed chemical speciation of gaseous, semivolatile, and particle-phase inorganic and organic compounds. Target analytes included metals, inorganic ions and gases, organic and elemental carbon, alkanes, alkenes, aromatic and aliphatic acids, aromatic hydrocarbons, polycyclic aromatic hydrocarbons (PAH), oxygenated PAH, nitrogenated PAH, isoprenoids, carbonyls, methoxyphenols, sugar derivatives, and sterols. The majority of the mass of material in the exposure atmospheres was gaseous nitrogen oxides and carbon monoxide, with lesser amounts of volatile organics and particle mass (PM) composed of carbon (approximately 90% of PM) and ions (approximately 10% of PM). Measured particle organic species accounted for about 10% of total organic particle mass and were mostly alkanes and aliphatic acids. Several of the components in the exposure atmosphere scaled in concentration with dilution but did not scale precisely with the dilution rate because of background from the rodents and scrubbed dilution air, interaction of animal derived emissions with diesel exhaust components, and day-to-day variability in the output of the engine. Rodent-derived ammonia reacted with exhaust to form secondary inorganic particles (at different rates dependent on dilution), and rodent respiration accounted for volatile organics (especially carbonyls and acids) in the same range as the diesel exhaust at the lowest exhaust exposure concentrations. Day-to-day variability in the engine output was implicated partially for differences of several components, including some of the particle bound organics. Though these observations have likely occurred in nearly all inhalation exposure atmospheres that contain complex mixtures of material, the speciations conducted here illustrate many of them for the first time.

Chronic inhalation exposure to mainstream cigarette smoke increases lung and nasal tumor incidence in rats.

An animal model of lung carcinogenicity induced by chronic inhalation of mainstream cigarette smoke would be useful for research on carcinogenic mechanisms, smoke composition-response relationships, co-carcinogenicity, and chemoprevention. A study was conducted to determine if chronic whole-body exposures of rats would significantly increase lung tumor incidence. Male and female F344 rats (n = 81 to 178/gender) were exposed whole-body 6 h/day, 5 days/week for up to 30 months to smoke from 1R3 research cigarettes diluted to 100 (LS) or 250 (HS) mg total particulate matter/m(3), or sham-exposed to clean air (C). Gross respiratory tract lesions and standard lung and nasal sections were evaluated by light microscopy. A slight reduction of survival suggested that the HS level was at the maximum tolerated dose as commonly defined. Cigarette smoke exposure significantly increased the incidences of non-neoplastic and neoplastic proliferative lung lesions in females, while nonsignificant increases were observed in males. The combined incidence of bronchioloalveolar adenomas and carcinomas in females were: HS = 14%; LS = 6%; and C = 0%. These incidences represented minima because only standard lung sections and gross lesions were evaluated. Mutations in codon 12 of the K-ras gene occurred in 4 of 23 (17%) tumors. Three mutations were G to A transitions and one was a G to T transversion. The incidence of neoplasia of the nasal cavity was significantly increased at the HS, but not the LS level in both males and females (HS = 6%, LS = 0.3%, C = 0.4% for combined genders). These results demonstrate that chronic whole-body exposure of rats to cigarette smoke can induce lung cancer.

Effects of cigarette smoke exposure and cessation on inflammatory cells and matrix metalloproteinase activity in mice.

B6C3F1 female mice were exposed to cigarette smoke (CS) (250 mg/m3 total particulate material) or filtered air (FA), 6 hours/day, 5 days/week, for 6, 7, or 10 weeks, or to CS for 6 weeks, then FA for 1 or 4 additional weeks. Exposure to CS increased macrophages, neutrophils, lymphocytes, and matrix metalloproteinase (MMP)-2 and MMP-9 content in bronchoalveolar lavage fluid. Partial recovery of most lavage parameters (except lymphocytes) was observed 1 week after cessation of CS exposure with further reductions after 4 weeks, but interstitial inflammation persisted longer. These results support a role for MMPs in CS-induced emphysema and indicate that smoking cessation allows restoration toward normal homeostasis.

A biphasic response to silica: I. Immunostimulation is restricted to the early stage of silicosis in lewis rats.

Inhalation of crystalline silica may lead to acute or chronic silicosis. Although chronic silicosis is associated with increased incidence/exacerbation of autoimmune disorders, the immunologic effects of chronic silicosis are not completely understood. In an animal model of chronic silicosis, Lewis rats were exposed to filtered air or silica (1.75 microm average particle size) at an exposure concentration of 6.2 mg/m(3), 6 h/d, 5 d/wk for 6 wk, and observed up to 27 wk after the exposure. Based on silica burden, lung histopathology, and immunologic changes, two distinct stages were identified in the development of chronic silicosis. Stage 1 (4-28 d after exposure) was characterized by silica deposition in various tissues, and augmented antibody and cellular immunity. Although bronchoalveolar lavage contained an increased number of activated macrophages, protein and lactate dehydrogenase levels were comparable to controls. In Stage 2 (>/= 10 wk), silica was localized in epithelioid macrophages, and T cell immunity had returned to normal, but the lavage fluids contained increased protein concentration and lactate dehydrogenase activity. Moreover, lungs from silica-treated animals contained neutrophils and lymphocytes, and exhibited granulomatous changes around the silica-containing epithelioid macrophages. Thus, in the early stages of silicosis, silica activates the immune system; however, the progression of lung granulomas does not depend on a continually activated adaptive immune system.

The uptake, distribution, metabolism, and excretion of methyl tertiary-butyl ether inhaled alone and in combination with gasoline vapor.

The purpose of these studies was to evaluate the tissue uptake, distribution, metabolism, and excretion of methyl tertiary-butyl ether (MTBE) in rats and to determine the effects of coinhalation of the volatile fraction of unleaded gasoline on these parameters. Male F344 rats were exposed nose-only once for 4 h to 4, 40, or 400 ppm 14C-MTBE and to 20 and 200 ppm of the light fraction of unleaded gasoline (LFG) containing 4 and 40 ppm 14C-MTBE, respectively. To evaluate the effects of repeated inhalation of LFG on the fate of inhaled MTBE, rats were exposed for 7 consecutive days to 20 and 200 ppm LFG followed on d 8 by exposure to LFG containing 14C-MTBE. Three subgroups of rats were included for evaluation of respiratory parameters, rates and routes of excretion, and tissue distribution and elimination. MTBE and its chief metabolite, tertiary-butyl alcohol, were quantitated in blood and kidney (immediately after exposure), and the major urinary metabolites, 2-hydroxyisobutyric acid and 2-methyl-1,2- propanediol, were identified and quantified in urine. Inhalation of MTBE alone or as a component of LFG had no concentration-dependent effect on respiratory minute volume. The initial body burdens (IBBs) of MTBE equivalents achieved after 4 h of exposure to MTBE did not increase linearly with exposure concentration. MTBE equivalents rapidly distributed to all tissues examined, with the largest percentages distributed to liver. Between 40 and 400 ppm, there was a significant reduction in percentage of the IBB present in the major organs examined, both immediately and 72 h after exposure. At 400 ppm, the elimination rates of MTBE equivalents from tissues changed significantly. Furthermore, at 400 ppm there was a significant decrease in the elimination half-time of volatile organic compounds (VOCs) in breath and a significant increase in the percentage of the IBB of MTBE equivalents eliminated as VOCs in breath. LFG coexposure significantly decreased the percentage of the MTBE equivalent IBBs in tissues and increased rates of elimination of MTBE equivalents. The study results indicate that the uptake and fate of inhaled MTBE are altered upon increasing exposure levels from 4 to 400 ppm, suggesting that toxic effects observed previously upon repeated inhalation of concentrations of 400 ppm or greater may not necessarily be linearly extrapolated to effects that might occur at lower concentrations. Furthermore, coexposure to LFG, whether acute or repeated, decreases tissue burdens of MTBE equivalents and enhances the elimination rate of MTBE and its metabolites, thereby potentially reducing the toxic effects of the MTBE compared to when it is inhaled alone.