Lung inflammation induced by endotoxin is enhanced in rats depleted of alveolar macrophages with aerosolized clodronate.

Clodronate liposomes were given to rats via intratracheal inhalation to investigate the importance of alveolar macrophages (AMs) in inhaled endotoxin-induced lung injury. When AM depletion was maximal (87% to 90%), rats were exposed to lipopolysaccharide (LPS) or saline. Neither clodronate nor saline liposomes induced an influx of neutrophils (PMNs) into the lungs. However, depleted LPS-exposed rats had 5- to 8-fold higher numbers of lavage PMNs and greater lavage cell reactive oxygen species release compared to undepleted rats. Although AM depletion by itself did not significantly increase inflammatory cytokine expression in lung tissue, LPS-induced message levels for interleukin (IL)-1alpha, IL-1beta, IL-6, and tumor necrosis factor (TNF)-alpha were approximately 2-fold higher in AM-depleted rats compared to undepleted rats. These results indicate that cells other than AMs can recruit inflammatory cells into the lungs during acute LPS-induced injury and that AMs play an important suppressive role in the innate pulmonary inflammatory response.

Translocation of inhaled ultrafine particles to the brain.

Ultrafine particles (UFP, particles <100 nm) are ubiquitous in ambient urban and indoor air from multiple sources and may contribute to adverse respiratory and cardiovascular effects of particulate matter (PM). Depending on their particle size, inhaled UFP are efficiently deposited in nasal, tracheobronchial, and alveolar regions due to diffusion. Our previous rat studies have shown that UFP can translocate to interstitial sites in the respiratory tract as well as to extrapulmonary organs such as liver within 4 to 24 h postexposure. There were also indications that the olfactory bulb of the brain was targeted. Our objective in this follow-up study, therefore, was to determine whether translocation of inhaled ultrafine solid particles to regions of the brain takes place, hypothesizing that UFP depositing on the olfactory mucosa of the nasal region will translocate along the olfactory nerve into the olfactory bulb. This should result in significant increases in that region on the days following the exposure as opposed to other areas of the central nervous system (CNS). We generated ultrafine elemental (13)C particles (CMD = 36 nm; GSD = 1.66) from [(13)C] graphite rods by electric spark discharge in an argon atmosphere at a concentration of 160 microg/m(3). Rats were exposed for 6 h, and lungs, cerebrum, cerebellum and olfactory bulbs were removed 1, 3, 5, and 7 days after exposure. (13)C concentrations were determined by isotope ratio mass spectroscopy and compared to background (13)C levels of sham-exposed controls (day 0). The background corrected pulmonary (13)C added as ultrafine (13)C particles on day 1 postexposure was 1.34 microg/lung. Lung (13)C concentration decreased from 1.39 microg/g (day 1) to 0.59 microg/g by 7 days postexposure. There was a significant and persistent increase in added (13)C in the olfactory bulb of 0.35 microg/g on day 1, which increased to 0.43 microg/g by day 7. Day 1 (13)C concentrations of cerebrum and cerebellum were also significantly increased but the increase was inconsistent, significant only on one additional day of the postexposure period, possibly reflecting translocation across the blood-brain barrier in certain brain regions. The increases in olfactory bulbs are consistent with earlier studies in nonhuman primates and rodents that demonstrated that intranasally instilled solid UFP translocate along axons of the olfactory nerve into the CNS. We conclude from our study that the CNS can be targeted by airborne solid ultrafine particles and that the most likely mechanism is from deposits on the olfactory mucosa of the nasopharyngeal region of the respiratory tract and subsequent translocation via the olfactory nerve. Depending on particle size, >50% of inhaled UFP can be depositing in the nasopharyngeal region during nasal breathing. Preliminary estimates from the present results show that approximately 20% of the UFP deposited on the olfactory mucosa of the rat can be translocated to the olfactory bulb. Such neuronal translocation constitutes an additional not generally recognized clearance pathway for inhaled solid UFP, whose significance for humans, however, still needs to be established. It could provide a portal of entry into the CNS for solid UFP, circumventing the tight blood-brain barrier. Whether this translocation of inhaled UFP can cause CNS effects needs to be determined in future studies.

On-road exposure to highway aerosols. 1. Aerosol and gas measurements.

On-road experiments were conducted to determine the sensitivities of rats to real-world aerosol. This article summarizes the on-road aerosol and gas measurements and provides background information for the companion paper on the rat exposures. Measurements were carried out over 10 days, 6 h/day, driving a route from Rochester to Buffalo. Aerosol instrumentation used in this study included two scanning mobility particle sizers (SMPS) to determine the aerosol size distribution from 10 to 300 nm, 2 stand-alone condensation particle counters to determine the total aerosol number concentration, and an electrical aerosol detector to determine the aerosol length concentration. A thermal denuder (TD) was used with one of the SMPS instruments to determine the size distribution of the non-volatile fraction. Filter samples were collected and analyzed for elemental carbon, and gas analyzers measured ambient levels of CO, CO(2), and NO. Average daily total aerosol number concentration ranged from 200,000 to 560,000 particles/cm(3). Past studies on urban highways have measured total number concentrations ranging between 10(4) and 10(6) particles/cm(3). The average daily NO concentration ranged from 0.10 to 0.24 ppm and the corresponding CO(2) concentration ranged from 400 to 420 ppm. The average daily geometric number mean particle size determined by the SMPS ranged from 15 to 20 nm. The TD reduced the average SMPS number concentration between 87 and 95% and the SMPS volume between 54 and 83%, suggesting that most of the particles consisted of volatile material. The TD also increased the geometric number mean diameter from 15 to 20 nm to 30 to 40 nm.

Efficient depletion of alveolar macrophages using intratracheally inhaled aerosols of liposome-encapsulated clodronate.

Rat alveolar macrophages (AMs) were depleted via intratracheal inhalation (ITIH) of clodronate-containing liposomes. AM depletion following ITIH delivery of clodronate liposomes was 33.2 +/- 14.2 on day 1, 88.1 +/- 6.2 on day 3, and 91.4 +/- 1.8 on day 4 relative to control rats given saline-containing liposomes. Almost all (approximately 99%) of the AMs remaining at the 3-day time point were peroxidase negative, suggesting that immature macrophages were not recruited from the circulation to replace those undergoing cell death on that day. Only 0.5% +/- 0.5% of bronchoalveolar lavage (BAL) cells were neutrophils at this time (normalized to controls). Whole-body inhalation did not induce as much AM depletion at 3 days (37.6% +/- 10.1%) and required larger amounts of liposome-encapsulated clodronate compared to ITIH. Intratracheal instillation (as opposed to inhalation) of clodronate liposomes produced a significant inflammatory response characterized by the influx of both polymorphonuclear neutrophils (PMNs) and macrophages. In subsequent pilot studies, the response to intratracheally instilled crystalline silica (75 microg) was found to be markedly reduced in rats depleted of AMs by the ITIH method. We conclude that ITIH of clodronate liposomes in rats is both efficient and useful for examining the role of AMs in pulmonary toxicology.

Formation of 8-oxo-7,8-dihydro-2'-deoxyguanosine in rat lung DNA following subchronic inhalation of carbon black.

Chronic high-dose inhalation of carbon black (CB) can produce carcinomas in rat lungs. The mechanisms underlying this response are uncertain. It has been hypothesized that chronic inflammation and cell proliferation may play a role in the development of tumors after high dose, long-term contact of the particles with lung epithelial cells. In this investigation, we analyzed the formation of a known mutagenic lesion [8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxo-dG)] in the lung DNA of rats following subchronic inhalation of CB (Printex-90 and Sterling V). Briefly, female Fischer 344 rats were exposed for 6 h/day, 5 days/week for 13 weeks to 1, 7, and 50 mg/m(3) of Printex-90 (16 nm; specific surface area 300 m(2)/g) and to 50 mg/m(3) of Sterling V CB (70 nm; surface area of 37 m(2)/g). The exposure concentration of Sterling V was selected to be equivalent in terms of retained mass in the lung to the high dose of Printex-90 at the end of exposure. However, in terms of retained particle surface area, the retained lung dose of Sterling V was equivalent to the mid-dose of Printex 90. This design allows comparison of results on the basis of retained particle mass as well as retained particle surface area between the two CB particles. The formation of 8-oxo-dG in the lung DNA was assessed using a reverse phase HPLC system coupled with UV and electrochemical (EC) detection. After 13 weeks of exposure, measurements were made on lung samples obtained at the end of the exposure and a 44-week recovery period in clean air. Lung burdens of CB were determined at both time points as well as differential cell populations from bronchoalveolar lavage fluid (BAL). The results indicate that lung particle overload was achieved after exposure to 7 and 50 mg/m(3) (Printex-90) and 50 mg/m3 (Sterling V) but not at 1 mg/m(3) (Printex-90). Consistent with these results, a significant increase (P < 0.05) in 8-oxo-dG induction was observed following 13 weeks of exposure to 50 mg/m(3) Printex-90 and at 7 and 50 mg/m(3) after the 44-week recovery period. Interestingly, no increase in 8-oxo-dG was observed for Sterling V CB at either time point despite lung particle overload. Although the retained mass dose of Sterling V at the end of exposure was even higher than for Printex 90 (50 mg/m(3) exposure group) (approximately 7.6 vs 4.8 mg), the surface area of the retained Sterling V was similar to that of the retained Printex 90 of the mid-dose exposure (7 mg/m(3)) (approximately 0.2 m(2) in both groups). Since both Sterling V (50 mg/m(3)) and Printex 90 (7 mg/m(3)) did not induce significant increases in 8-oxo-dG in the lung at the end of the 13-week exposure, this finding indicates that a retained large particle mass is not always correlated with similar adverse effects but that particle surface area is a better dose parameter. The lower effect per unit mass dose seen with Sterling V is consistent with earlier studies showing that particle surface area of low toxicity particles is a more appropriate dosemetric for induction of inflammation in the lungs than particle mass (Oberdörster et al., 1994, 2001; Brown et al. 2001; Donaldson et al., 2002). An increase (p < 0.05) in lung lavage neutrophils was observed at 7 mg/m(3) (Printex-90) and 50 mg/m(3) (Printex-90 and Sterling V) at the 13-week exposure period and again at 50 mg/m(3) (Printex-90 and Sterling V, 44-week recovery period). Our current findings suggest that prolonged, high-dose exposure to CB can promote oxidative DNA damage that is consistent with the hypothesis that inflammatory cell-derived oxidants may play a role in the pathogenesis of rat lung tumors following long-term high-dose exposure to CB in rats.

Pulmonary effects induced by ultrafine PTFE particles.

PTFE (polytetrafluoroethylene) fumes consisting of large numbers of ultrafine (uf) particles and low concentrations of gas-phase compounds can cause severe acute lung injury. Our studies were designed to test three hypotheses: (i) uf PTFE fume particles are causally involved in the induction of acute lung injury, (ii) uf PTFE elicit greater pulmonary effects than larger sized PTFE accumulation mode particles, and (iii) preexposure to the uf PTFE fume particles will induce tolerance. We used uf Teflon (PTFE) fumes (count median particle size approximately 16 nm) generated by heating PTFE in a tube furnace to 486 degrees C to evaluate principles of ultrafine particle toxicity. Teflon fumes at ultrafine particle concentrations of 50 microg/m(3) were extremely toxic to rats when inhaled for only 15 min. We found that when generated in argon, the ultrafine Teflon particles alone are not toxic at these exposure conditions; neither were Teflon fume gas-phase constituents when generated in air. Only the combination of both phases when generated in air caused high toxicity, suggesting either the existence of radicals on the surface or a carrier mechanism of the ultrafine particles for adsorbed gas compounds. Aging of the fresh Teflon fumes for 3.5 min led to a predicted coagulation to >100 nm particles which no longer caused toxicity in exposed animals. This result is consistent with a greater toxicity of ultrafine particles compared to accumulation mode particles, although changes in particle surface chemistry during the aging process may have contributed to the diminished toxicity. Furthermore, the pulmonary toxicity of the ultrafine Teflon fumes could be prevented by adapting the animals with short 5-min exposures on 3 days prior to a 15-min exposure. Messages encoding antioxidants and chemokines were increased substantially in nonadapted animals, yet were unaltered in adapted animals. This study shows the importance of preexposure history for the susceptibility to acute ultrafine particle effects.

Pulmonary chemokine and mutagenic responses in rats after subchronic inhalation of amorphous and crystalline silica.

Chronic inhalation of crystalline silica can produce lung tumors in rats whereas this has not been shown for amorphous silica. At present the mechanisms underlying this rat lung tumor response are unknown, although a significant role for chronic inflammation and cell proliferation has been postulated. To examine the processes that may contribute to the development of rat lung tumors after silica exposure, we characterized the effects of subchronic inhalation of amorphous and crystalline silica in rats. Rats were exposed for 6 h/day, on 5 days/week, for up to 13 weeks to 3 mg/m(3) crystalline or 50 mg/m(3) amorphous silica. The effects on the lung were characterized after 6.5 and 13 weeks of exposure as well as after 3 and 8 months of recovery. Exposure concentrations were selected to induce high pulmonary inflammatory-cell responses by both compounds. Endpoints characterized after silica exposure included mutation in the HPRT gene of isolated alveolar cells in an ex vivo assay, changes in bronchoalveolar lavage fluid markers of cellular and biochemical lung injury and inflammation, expression of mRNA for the chemokine MIP-2, and detection of oxidative DNA damage. Lung burdens of silica were also determined. After 13 weeks of exposure, lavage neutrophils were increased from 0.26% (controls) to 47 and 55% of total lavaged cells for crystalline and amorphous silica, with significantly greater lavage neutrophil numbers after amorphous silica (9.3 x 10(7) PMNs) compared to crystalline silica (6.5 x 10(7) PMNs). Lung burdens were 819 and 882 microg for crystalline and amorphous silica, respectively. BAL fluid levels of LDH as an indicator of cytotoxicity were twice as high for amorphous silica compared to those of crystalline silica, at the end of exposure. All parameters remained increased for crystalline silica and decreased rapidly for amorphous silica in the 8-month recovery period. Increased MIP-2 expression was observed at the end of the exposure period for both amorphous and crystalline silica. After 8 months of recovery, those markers remained elevated in crystalline silica-exposed rats, whereas amorphous silica-exposed rats were not significantly different from controls. A significant increase in HPRT mutation frequency in alveolar epithelial cells was detected immediately after 13 weeks of exposure to crystalline, but not to amorphous silica. A significant increase in TUNEL staining was detected in macrophages and terminal bronchiolar epithelial cells of amorphous silica-exposed rats at the end of the exposure period; however, crystalline silica produced far less staining. The observation that genotoxic effects in alveolar epithelial cells occurred only after crystalline but not amorphous silica exposure, despite a high degree of inflammatory-cell response after subchronic exposure to both types of silica, suggests that in addition to an inflammatory response, particle biopersistence, solubility, and direct or indirect epithelial cell cytotoxicity may be key factors for the induction of either mutagenic events or target cell death.

Antioxidant and inflammatory response after acute nitrogen dioxide and ozone exposures in C57Bl/6 mice.

Ozone (O(3)) and nitrogen dioxide (NO(2)) are highly reactive and toxic oxidant pollutants. The objective of this study is to compare chemokine, cytokine, and antioxidant changes elicited by acute exposures of O(3) and NO(2) in a genetically sensitive mouse. Eight-week-old C57Bl/6J mice were exposed to 1 or 2.5 ppm ozone or 15 or 30 ppm NO(2) for 4 or 24 h. Changes in mRNA abundance in lung were assayed by slot blot and ribonuclease protection assay (RPA). Messages encoding metallothionein (Mt), heme oxygenase I (HO-I), and inducible nitric oxide synthase (iNOS) demonstrated increased message abundance after 4 and 24 h of exposure to either O(3) or NO(2). Furthermore, increases in message abundance were of a similar magnitude for O(3) and NO(2). Messages encoding eotaxin, macrophage inflammatory protein (MIP)-1alpha, and MIP-2 were elevated after 4 and 24 h of exposure to 1 ppm ozone. Interleukin-6 was elevated after 4 h of exposure to ozone. After 4 h of 2.5 ppm ozone exposure, increased mRNAs of eotaxin, MIP-1alpha, MIP-2, Mt, HO-I, and iNOS were elevated to a higher magnitude than were detected after 1 ppm ozone. Monocyte chemoattractant protein (MCP-1) was elevated following 15 ppm NO(2) exposure. After 4 h of 30 ppm NO(2) exposure, messages encoding eotaxin, MIP-1alpha, MIP-2, and MCP-1 were elevated to levels similar to those detected after ozone exposure. Our results demonstrate a similar antioxidant and chemokine response during both O(3) and NO(2) exposure. Induction of these messages is associated with the duration and concentration of exposure. These studies suggest that these gases exert toxic action through a similar mechanism.

Newborn mice differ from adult mice in chemokine and cytokine expression to ozone, but not to endotoxin.

Neonatal animals of some mammalian species are more tolerant to several pulmonary oxidative stress-inducing toxicants than adults. Our initial studies during hyperoxic injury demonstrated a rapid chemokine and cytokine response early in the development of injury in newborn mice, whereas adult mice demonstrated little alteration in cytokine abundance until lethality was imminent. Our hypothesis is that altered response between newborn and adult mice is associated with differential cell injury, rather than alterations in the regulation of the inflammatory response. To test this hypothesis we utilized two distinct models of inducing pulmonary toxicity: ozone (O(3)), which causes epithelial cell injury, and endotoxin, which causes pulmonary inflammation independent of direct epithelial cell injury. C57Bl/6J mice (36 h or 8 wk old) were exposed to O(3) at 1 or 2.5 ppm for 4, 20, or 24 h or to a 10-min inhalation of 10 ng endotoxin per mouse (estimated deposited dose) and were examined 2, 6, or 24 h postexposure. Adult mice displayed increased sensitivity to O(3), as demonstrated by increased abundance of mRNAs encoding eotaxin, macrophage inflammatory protein (MIP)-1alpha, MIP-2, interleukin (IL)-6, and metallothionein (Mt). In newborn mice, only Mt was increased after 4 h of exposure. In contrast, newborn and adult mice responded similarly at 2 h post endotoxin exposure, inducing messages encoding tumor necrosis factor (TNF)-alpha, eotaxin, MIP-1alpha, MIP-1beta, MIP-2, interferon inducible protein (IP)-10, and monocyte chemoattractant protein (MCP)-1. Furthermore, interleukin-6 (IL-6) was increased in adults but not newborns. Similar chemokine and cytokine responses of newborn and adult mice in response to an agent not causing epithelial injury (endotoxin) suggest that altered inflammatory control observed between newborn and adult mice following O(3) exposure is secondary to epithelial cell injury.

Induction of adaptation to inhaled lipopolysaccharide in young and old rats and mice.

Lipopolysaccharide (LPS) is a component of the gram-negative bacterial cell wall that is known to activate inflammatory cells and enhance the production of inflammatory mediators in the lung. As it is a ubiquitous compound, inhalation exposure is highly likely in the human environment. Adaptation is a phenomenon by which a previous exposure results in improved survival or reduced injury as compared to a single exposure alone. We hypothesized that the basic proinflammatory effects of LPS in the lung could result in the development of adaptation in animals. Based on evidence of age- and species-related differences in lung injury, we used an acute lung injury model with inhaled LPS to compare the development of adaptation in young and old Fisher 344 rats and C57Bl/6J mice. Animals were exposed to low-dose (predicted lung deposition approximately 20 ng in rats and approximately 5 ng in mice) LPS aerosols for 10 min on 3 consecutive days; on day 4, a high dose (rats approximately 200 ng; mice approximately 25 ng) was delivered. Another group of animals received only the high LPS dose on day 4, whereas controls were unexposed. Twenty-four hours after the last exposure, cellular and inflammatory parameters in bronchoalveolar lavage (BAL) were determined. An adaptive response was found in both rats and mice. Adapted animals showed significantly fewer BAL neutrophils compared to nonadapted ones; there was also a significantly lower release of oxidants from phorbol methyl ester-stimulated BAL cells from adapted compared to nonadapted animals, which, in turn, showed a greater response than controls. Furthermore, studies in old animals (21 mo of age) showed that adaptation also occurs in this age group. The adaptive response is clear in old mice; in rats, there is greater variability in the response, but an adaptive trend is apparent. Therefore, we have demonstrated that inhaled low-dose LPS can induce adaptation to subsequent higher doses, much as has been shown for other toxicants that induce oxidative lung injury.

Ultrafine Particle Concentrations in a Hospital.

Ultrafine particles (UFP) may contribute to the morbidity and mortality associated with exposure to ambient particles, but few data are available on ultrafine particle numbers in indoor air, where susceptible subjects spend most of their time. We measured particle number, UFP size distribution, and total suspended particulate (JSP) mass in three locations: (I) a medical floor in a large tertiary care hospital, (2) outdoor air above a construction site outside the hospital, and (3) an environmental exposure chamber with purification of intake air. Mass and number concentrations were recorded continuously in each location over 70-110 h. Mean ± SD particle (p) numbers were 3.63 ± 1.l5 } 10(3) p/cm(3) in the hospital, 3.05 ± 6.65 } 10(4) p/cm(3) outside, and 5.86 ± 2.11 } 10(2) p/cm(3) in the environmental chamber. In the hospital, particle number and mass declined during the evening hours when the unit was less active, with the particle number as low as 1.15 } 10(3) p/cm(3). Particle numbers peaked (2.78 } 10(4) p/cm(3)) in the morning hours when activity on the unit was the most intense. "Spikes" in fine particle number were often not accompanied by increases in TSP mass. In the hospital, a distinct population of ultrafine particles (median diameter approximately 23 nm) was observed during the lunch hour, suggesting a change in particle source during this time. Outdoor fine particle numbers above the construction site were highly variable, reaching peaks of greater than 1.7 } 10(6) p/cm(3). These data suggest that, in the indoor environment, particle numbers and size distribution vary with intensity and type of local activity, and significant peaks in particle number are not detected with daily averages. Monitoring of particle mass may be an inaccurate measure of exposure to ultrafine particles indoors.

Pulmonary inflammatory response to inhaled ultrafine particles is modified by age, ozone exposure, and bacterial toxin.

Epidemiological studies demonstrate associations between increasing levels of ambient particles and morbidity in the elderly with cardiopulmonary disease. Such findings have been challenged partly because particles may not act alone to cause these effects. We hypothesized that carbonaceous ambient ultrafine particles and ozone can act together to induce greater oxidative stress and inflammation in the lung than when administered alone and that these effects would be amplified in the compromised, aging lung. Two models of a compromised lung were used: endotoxin priming and old-age emphysema (TSK mice). Young (10 wk) and old (22 mo) male F344 rats and male TSK mice (14-17 mo) were exposed to ultrafine carbon particles (count median diameter 25 nm, 110 micrograms/m3) and to ozone (1 ppm) alone and in combination for 6 h. Inhalation of low-dose endotoxin (70 and 7.5 units estimated alveolar deposited dose in rats and mice, respectively) was used to model respiratory-tract infection. Cellular and biochemical lavage parameters and oxidant release from lung lavage cells were assessed 24 h after exposure. Inflammatory cell influx into the alveolar space was observed for both species and age groups: The combination of inhaled ultrafine carbon and ozone after endotoxin priming resulted in the greatest increase in lavage-fluid neutrophils. In general, the unstimulated and stimulated release of reactive oxygen species (ROS) from lavage inflammatory cells correlated well with the neutrophil response. There were significant effects of carbon particles as well as a consistent interaction between carbon and ozone as determined by analysis of variance (ANOVA). However, this interaction was in the opposite direction in young rats versus old rats and old TSK mice: Carbon and ozone interacted such that ROS activity was depressed in young rats, whereas it was enhanced in old rats and old TSK mice, indicating age-dependent functional differences in elicited pulmonary inflammatory cells. These results demonstrate that ultrafine carbonaceous particles inhaled for short periods of time can induce significant pulmonary inflammation and oxidative stress that are modified by age, copollutants, and a compromised respiratory tract.

Acute pulmonary effects of ultrafine particles in rats and mice.

Ambient fine particles consist of ultrafine particles (< 100 nm) and accumulation-mode particles (approximately 100 to 1,000 nm). Our hypothesis that ultrafine particles can have adverse effects in humans is based on results of our earlier studies with particles of both sizes and on the finding that urban ultrafine particles can reach mass concentrations of 40 to 50 micrograms/m3, equivalent to number concentrations of 3 to 4 x 10(5) particles/cm3. The objectives of the exploratory studies reported here were to (1) evaluate pulmonary effects induced in rats and mice by ultrafine particles of known high toxicity (although not occurring in the ambient atmosphere) in order to obtain information on principles of ultrafine particle toxicology; (2) characterize the generation and coagulation behavior of ultrafine particles that are relevant for urban air; (3) study the influence of animals' age and disease status; and (4) evaluate copollutants as modifying factors. We used ultrafine Teflon (polytetrafluoroethylene [PTFE]*) fumes (count median diameter [CMD] approximately 18 nm) generated by heating Teflon in a tube furnace to 486 degrees C to evaluate principles of ultrafine particle toxicity that might be helpful in understanding potential effects of ambient ultrafine particles. Teflon fumes at ultrafine particle concentrations of approximately 50 micrograms/m3 are extremely toxic to rats when inhaled for only 15 minutes. We found that neither the ultrafine Teflon particles alone when generated in argon nor the Teflon fume gas-phase constituents when generated in air were toxic after 25 minutes of exposure. Only the combination of both phases when generated in air caused high toxicity, suggesting the existence of either radicals on the particle surface or a carrier mechanism of the ultrafine particles for adsorbed gas-phase compounds. We also found rapid translocation of the ultrafine Teflon particles across the epithelium after their deposition, which appears to be an important difference from the behavior of larger particles. Furthermore, the pulmonary toxicity of the ultrafine Teflon fumes could be prevented by adapting the animals with short 5-minute exposures on 3 days prior to a 15-minute exposure. This shows the importance of preexposure history in susceptibility to acute effects of ultrafine particles. Aging of the fresh Teflon fumes for 3.5 minutes led to a predicted coagulation resulting in particles greater than 100 nm that no longer caused toxicity in exposed animals. This result is consistent with greater toxicity of ultrafine particles compared with accumulation-mode particles. When establishing dose-response relationships for intratracheally instilled titanium dioxide (TiO2) particles of the size of the urban ultrafine particles (20 nm) and of the urban accumulation-mode particles (250 nm), we observed significantly greater pulmonary inflammatory response to ultrafine TiO2 in rats and mice. The greater toxicity of the ultrafine TiO2 particles correlated well with their greater surface area per mass. Ultrafine particles of carbon, platinum, iron, iron oxide, vanadium, and vanadium oxide were generated by electric spark discharge and characterized to obtain particles of environmental relevance for study. The CMD of the ultrafine carbon particles was approximately 26 nm, and that of the metal particles was 15 to 20 nm, with geometric standard deviations (GSDs) of 1.4 to 1.7. For ultrafine carbon particles, approximately 100 micrograms/m3 is equivalent to 12 x 10(6) particles/cm3. Homogeneous coagulation of these ultrafine particles in an animal exposure chamber occurred rapidly at 1 x 10(7) particles/cm3, so that particles quickly grew to sizes greater than 100 nm. Thus, controlled aging of ultrafine carbon particles allowed the generation of accumulation-mode carbon particles (due to coagulation growth) for use in comparative toxicity studies. We also developed a technique to generate ultrafine particles consisting of the stable isotope 13C by using 13C-graphite electrodes made in our laboratory from amorphous 13C powder. These particles are particularly useful tools for determining deposition efficiencies of ultrafine carbon particles in the respiratory tracts of laboratory animals and the translocation of particles to extrapulmonary sites. For compromised animals, we used acute and chronic pulmonary emphysema; a low-dose endotoxin inhalation aimed at priming target cells in the lung was also developed. Other modifying factors were age and copollutant (ozone) exposure. Exposure concentrations of the generated ultrafine particles for acute rodent inhalation studies were selected on the basis of lung doses predicted to occur in people inhaling approximately 50 micrograms/m3 urban ultrafine particles. Concentrations that achieved the same predicted lung burden per unit alveolar surface were used in rodents. (ABSTRACT TRUNCATED)

Normal function and lack of fibronectin accumulation in kidneys of Clara cell secretory protein/uteroglobin deficient mice.

Clara cell secretory protein (CCSP), also known as uteroglobin (Ug), is a 16-kDa homodimeric protein of unknown function. Within rodent species, CCSP is expressed predominantly by nonciliated Clara cells that line conducting airways of the lung. To investigate in vivo functions for CCSP, we established mice homozygous for a null allele of the CCSP gene (CCSP-/-). We previously showed no overt phenotypic consequences associated with CCSP deficiency when CCSP-/- mice are maintained in the absence of environmental stress. However, CCSP-/- mice show an oxidant-sensitive phenotype that cannot be attributed to alterations in the inflammatory response when challenged by inhaled oxidant gases. The current study was undertaken to determine whether CCSP deficiency results in pathological changes to the kidney. This study was prompted by the recent description of severe systemic disease and kidney fibrosis/dysfunction in an independent line of CCSP-deficient mice, termed Ug-/- (Zhang et al, Science 276:1408-1412, 1997). CCSP-/- mice show normal growth and reproductive performance when maintained in two independent genetic backgrounds, inbred 129 and congenic C57BL/6. Strain 129 CCSP-/- mice have normal kidney function, as assessed by urinary glucose, lactate dehydrogenase, and glomerular filtration rate; they show no kidney fibrosis or abnormalities in fibronectin accumulation and no histological abnormalities in proximal convoluted tubules or glomeruli at either light or electron microscopic levels. CCSP deficiency is associated with mild proteinurea involving a modest increase in mouse major urinary protein-1. We conclude that CCSP (Ug) deficiency, per se, is not the cause of severe renal pathology and systemic disease reported for Ug-/- mice.

Quantitative exposure of humans to an octamethylcyclotetrasiloxane (D4) vapor.

There is potential for human exposure to cyclic siloxanes by the respiratory route. To determine the pharmacokinetics of octamethylcyclotetrasiloxane (D4), a material commonly found in personal care products, the respiratory intake and uptake of D4 were measured in 12 healthy volunteers (25-49 years) on two occasions. Subjects inhaled 10 ppm D4 (122 micrograms/liter) or air (control) during a 1-h exposure via a mouthpiece in a double-blind, randomized fashion. Inspiratory and expiratory D4 concentrations were continuously measured. Exhaled air and plasma D4 levels were measured before, during, and after exposures. Individual D4 uptakes were measured under steady-state conditions during three rest periods (10, 20, and 10 min, respectively) alternating with two 10-min exercise periods. Mean D4 intake was 137 +/- 25 mg (SD) and the mean deposition efficiency was equivalent to 0.74/(1 + 0.45 VE), where VE is the minute ventilation. No changes in lung function were induced by the D4 vapor. Plasma measurements of D4 gave a mean peak value of 79 +/- 5 ng/g (SEM) and indicated a rapid nonlinear blood clearance. Using lung volume and respiratory surface area estimates based on functional residual capacity measurements, we developed a model and determined that the effective mass transfer coefficient for D4 was 5.7 x 10(-5) cm/s from lung air to blood. In an additional eight subjects, we compared D4 deposition with mouthpiece and nasal breathing at resting ventilations. For these individuals, mean deposition was similar for the two exposure protocols, averaging 12% after correction for exposure system losses. These are the first data describing the intake and absorption of D4 and they should contribute to a meaningful safety assessment of the compound.

Pulmonary cytokine and chemokine mRNA levels after inhalation of lipopolysaccharide in C57BL/6 mice.

Inhaled endotoxin (lipopolysaccharide, LPS) can induce acute lung injury and at high doses may lead to respiratory distress syndrome. Using a mouse model of acute lung inflammation induced by inhalation of low doses of LPS we examined the kinetics of chemokine, proinflammatory cytokine, and metallothionein. Eight-week-old C57BL/6 mice were dosed for 10 min with LPS, resulting in an estimated alveolar dose of < 10 ng LPS/mouse, and euthanized 2,6, or 24 h postexposure. Analysis of bronchoalveolar lavage fluid demonstrated increased polymorphonuclear neutrophils (PMNs) of 6.94, 32.7, and 38.8% after 2, 6, and 24 h, respectively. Examination of proinflammatory cytokine, chemokine, and Mt mRNA in the lung revealed increases for messages encoding IL-1 alpha, IL-1 beta, IL-6, IFN-gamma, TNF alpha, Eotaxin, MIP-1 alpha, MIP-1 beta, MIP-2, Mt, and IP-10, while messages encoding IL-12, IL-10, IFN-beta, Ltn, MCP-1, TGF beta 1 + 2, and RANTES were unchanged from those of sham-exposed mice 2 h postexposure. By 6 h most messages had returned to near control levels. Comparison to 5 mg/kg body weight intraperitoneal injection and 5 micrograms/mouse intratracheal instillation 2 h postexposure demonstrated similar message responses. Our results demonstrate that low levels of LPS exposure by inhalation induce a strong PMN response and a selective cytokine response in the lung, supporting the hypothesis that PMNs may regulate inflammatory processes via cytokine and chemokine response.

Intratracheal instillation versus intratracheal-inhalation of tracer particles for measuring lung clearance function.

Effective elimination of particles deposited in the respiratory tract is an important defense function to protect the organism from potentially adverse effects of inhaled particles. Delivery of radioactively labeled tracer particles and subsequent measurement in vivo of their retention in different regions of the respiratory tract provides an adequate method for characterizing this defensive function. However, the delivery of such tracer particles by inhalation may result in some external contamination of the animals and requires specific protective measures while working with radioactive aerosols. In this study, 85Sr-labeled tracer particles (3 microns) were administered to the lower respiratory tract of rats by intratracheal inhalation to avoid external contamination, and also by intratracheal instillation in order to compare the 2 technique with respect to their suitability for measuring normal and impaired particle clearance rates. It was postulated that particle clearance function in the alveolar region can be determined equally well with intratracheally instilled particles despite their uneven distribution in the lung. For both techniques, rats were anesthesized with halothane and the particles were administered via oral intubation. Retention in the lower respiratory tract of about 30 micrograms (inhalation) and 6 micrograms (instillation) of the administered particles was followed over a period of 180 days by external counting of lung 85Sr-activity in a collimated detection system. To impair alveolar particle clearance rates, groups of rats were subjected to 12 weeks of inhalation exposure prior to delivery of the tracer particles as follows: (1) sham-exposed control; (2) pigment-grade TiO2 particles to induce lung overload: (3) ultrafine TiO2 particles: (4) crystalline SiO2 particles (cristobalite). The following results were obtained: The long-term retention half-times (T1/2) of the tracer particles reflecting alveolar clearance consistently showed the same ranking of the treatment groups whether measured after intratracheal inhalation or instillation. Control values were 66 and 72 days, respectively, and significantly prolonged long-term clearance was measured by both methods for pigment-grade TiO2 (117 and 99 days), ultrafine TiO2 (541 and 600 days) and SiO2 (1901 and 1368 days). Comparison of these values between the two modes of administration of tracer particles showed no significant differences. In contrast, the short-term T1/2 (mucociliary clearance) of the intratracheally instilled tracer particles in the different treatment groups were variable and did not accurately reflect particle clearance from the conducting airways. However, short-term T1/2 after intratracheal inhalation of tracer particles were consistent with fast conducting airway clearance, and mucociliary clearance appears to be stimulated when alveolar clearance is significantly impaired due to particle overload or to effects of cytotoxic particles. The results suggest that intratracheal instillation of a low dose (< or = 10 micrograms) of tracer particles in the rat provides an adequate method for reliably determining effects of inhaled toxicants on alveolar particle clearance function. Further, intratracheal inhalation of tracer particles is useful for measuring both short-term (mucociliary) and long-term (alveolar) particle clearance rates in the lower respiratory tract of the rat.

Perinatal methanol exposure in the rat. I. Blood methanol concentration and neural cell adhesion molecules.

Although the acute toxicity of methanol is well documented, few studies have addressed the consequences of perinatal exposures to the low concentrations that are expected to arise from its proposed use as a component of automobile fuel. This report describes the general research design of a series of studies, the effects of methanol exposures on blood concentrations in dams and neonates, and indices of brain development. Four cohorts of Long-Evans pregnant rats, each cohort consisting of an exposure (n = 12) and a control (n = 12) group, were exposed whole-body to 4500 ppm methanol vapor or air for 6 hr daily beginning on Gestation Day 6. Both dams and pups were then exposed through Postnatal Day 21 (PND 21). Blood methanol concentrations determined by gas chromatography from samples obtained immediately following a 6-hr exposure reached approximately 500-800 micrograms/ml in the dams during gestation and lactation. Average concentrations for pups attained levels about twice those of the dams. Selected offspring from Cohort 4 were exposed for one additional 6-hr session at ages that extended out to PND 52. Regression analyses showed that the blood methanol concentrations of the pups declined until about PND 48, at which time their levels approximated those of their dams. Such pharmacokinetic differences might increase the risks posed to developing organisms. Light-microscopic analysis showed no significant abnormalities in the brains of the methanol-treated animals. However, assays of neural cell adhesion molecules (NCAMs) in brains of pups sacrificed on PND 4 showed staining for both the 140 and the 180 kDa isoforms to be less intense in the cerebellum of exposed animals. NCAM differences were not apparent in animals sacrificed 15 months after their final exposure.

Characterization of the early pulmonary inflammatory response associated with PTFE fume exposure.

Heating of polytetrafluoroethylene (PTFE) has been described to release fumes containing ultrafine particles (approximately 18 nm diam). These fumes can be highly toxic in the respiratory tract inducing extensive pulmonary edema with hemorrhagic inflammation. Fischer-344 rats were exposed to PTFE fumes generated by temperatures ranging from 450 to 460 degrees C for 15 min at an exposure concentration of 5 x 10(5) particles/cm3, equivalent to approximately 50 micrograms/m3. Responses were examined 4 hr post-treatment when these rats demonstrated 60-85% neutrophils (PMNs) in their lung lavage. Increases in abundance for messages encoding the antioxidants manganese superoxide dismutase and metallothionein (MT) increased 15- and 40-fold, respectively. For messages encoding the pro- and anti-inflammatory cytokines: inducible nitric oxide synthase, interleukin 1 alpha, 1 beta, and 6 (IL-1 alpha, IL-1 beta, and IL-6), macrophage inflammatory protein-2, and tumor necrosis factor-alpha (TNF alpha) increases of 5-, 5-, 10-, 40-, 40-, and 15-fold were present. Vascular endothelial growth factor, which may play a role in the integrity of the endothelial barrier, was decreased to 20% of controls. In situ sections were hybridized with 33P cRNA probes encoding IL-6, MT, surfactant protein C, and TNF alpha. Increased mRNA abundance for MT and IL-6 was expressed around all airways and interstitial regions with MT and IL-6 demonstrating similar spatial distribution. Large numbers of activated PMNs expressed IL-6, MT, and TNF alpha. Additionally, pulmonary macrophages and epithelial cells were actively involved. These observations support the notion that PTFE fumes containing ultrafine particles initiate a severe inflammatory response at low inhaled particle mass concentrations, which is suggestive of an oxidative injury. Furthermore, PMNs may actively regulate the inflammatory process through cytokine and antioxidant expression.

Human performance during exposure to toluene.

PURPOSE: The purpose of this research was to examine the effects of inhalation of toluene on respiratory function and neuropsychological performance of humans. METHODS: We exposed six healthy adults to 100 ppm toluene or air (control) for 6 h, in a double-blind, randomized fashion, with exposures separated by at least 14 d and including 30 min of exercise at a level that quadrupled minute ventilation. Blood and exhaled air toluene levels were measured before, during, immediately, and 1 and 2 h post-exposure. Lung function was measured before and immediately after exposure. Three repetitions of two computerized neuropsychological tests were performed, including a brief standard neuropsychological battery (ANAM) and a 1-h complex performance test (SYNWORK). Statistical analysis of the psychological data was conducted as a repeated measures ANOVA. FINDINGS: Following exercise, the mean blood and exhaled air toluene levels averaged 1.5 micrograms and 28 ppm, respectively. Lung function was unchanged post-exposure. On the SYNWORK test, the Composite score obtained over time during toluene exposure was lower than that during room air (F = 29.20, p = 0.005), with the score from the final hour reduced by 10%. On standard neuropsychological tests, latency but not accuracy proved the sensitive measure for five of the seven subtests presented. CONCLUSIONS: Performance of complex tests and response time to simple brief tests can be disrupted by toluene inhalation at 100 ppm. Differences in performance between air and toluene conditions were greatest after exercise, indicating that physical activity may enhance the response to volatile organic solvents.