Faculty Bibliography

While ambient acid aerosols are considered a potential respiratory health hazard, the mechanism by which they induce responses in the lungs is not known. Attempts to ascertain these mechanisms using inhalation exposures are complicated by a number of technical difficulties, chief among which are neutralization of inhaled acids by endogenous ammonia and variations in deposition with inhaled particle size. To control for these variables, a novel in vitro exposure system allowing experimental evaluation of factors which influence biologic responses to acid sulfate particles was developed. The system consists of two subunits, a generation/delivery component and a cell exposure component. Sulfuric acid aerosols are generated by nebulizing dilute acid solutions. Particles larger than a specified size of interest (based upon the specific exposure conditions desired) are removed, and particles at the desired size and mass concentration are uniformly delivered onto a target cell monolayer. The system is capable of delivering acid particles larger than 0.7 micron (mass median diameter), yet at constant particle mass concentrations. This paper describes the design of the exposure system and its performance characteristics and presents initial results of some biological responses obtained using it. In conjunction with inhalation studies, this exposure system may provide additional insights into mechanisms by which acid aerosols adversely affect the respiratory tract and into the physical characteristics of acid particles which modulate toxicity

Examining interspecies differences in response to ambient pollutants is an essential component of risk assessment. The potential hazard to public health from the inhalation of acid sulfate aerosols is of current concern. A significant biological target is the pulmonary macrophage, which provides a primary defense of the respiratory region of the lungs. One essential function of these cells is phagocytosis of particles. This study assessed the effects of acidic environments on the phagocytic activity of pulmonary macrophages obtained by lavage from humans and three species of laboratory animals commonly used in acid aerosol toxicology studies, namely, rats, rabbits, and guinea pigs. Cells were incubated with polystyrene latex particles in media acidified by addition of sulfuric acid. The percentage of cells which were phagocytic, as well as the relative number of particles ingested by these cells, was found to decrease with increasing acidity for all species. The ranking of response in order of decreasing sensitivity to acidic challenge was as follows: guinea pig > rat > rabbit > human

Occupational exposure to freshly formed zinc oxide (ZnO) particles (less than 1.0 micron aerodynamic diameter) produces a well-characterized response known as metal fume fever. An 8-hr threshold limit value (TLV) of 5 mg/m3 has been established to prevent adverse health effects because of exposure to ZnO fumes. Because animal toxicity studies have demonstrated pulmonary effects near the current TLV, the present study examined the time course and dose-response of the pulmonary injury produced by inhaled ZnO in guinea pigs, rats, rabbits, and human volunteers. The test animals were exposed to 0, 2.5, or 5.0 mg/m3 ZnO for up to 3 hr and their lungs lavaged. Both the lavage fluid and recovered cells were examined for evidence of inflammation or altered cell function. The lavage fluid from guinea pigs and rats exposed to 5 mg/m3 had significant increases in total cells, lactate dehydrogenase, beta-glucuronidase, and protein content. These changes were greatest 24 hr after exposure. Guinea pig alveolar macrophage function was depressed as evidenced by in vitro phagocytosis of opsonized latex beads. Significant changes in lavage fluid parameters were also observed in guinea pigs and rats exposed to 2.5 mg/m3 ZnO. In contrast, rabbits showed no increase in biochemical or cellular parameters following a 2-hr exposure to 5 mg/m3 ZnO. Differences in total lung burden of ZnO, as determined in additional animals by atomic absorption spectroscopy, appeared to account for the observed differences in species responses. Although the lungs of guinea pigs and rats retained approximately 20% and 12% of the inhaled dose, respectively, rabbits retained only 5%.(ABSTRACT TRUNCATED AT 250 WORDS)

Studies examining effects of air pollutants often use single compounds, while 'real world' exposures are to more than one chemical. Thus, it is necessary to assess responses following inhalation of chemical mixtures. Rabbits were exposed for 3 hr to sulfuric acid aerosol at 0, 50, 75, or 125 micrograms/m3 in conjunction with ozone at 0, 0.1, 0.3, or 0.6 ppm, following which broncho-pulmonary lavage was performed. Various pulmonary response endpoints related to general cytotoxicity and macrophage function were examined. In addition, a goal of the study was to define an improved approach to the analysis of data sets involving binary pollutant mixtures. Results were evaluated using analysis of variance with multiple linear contrasts to determine the significance of any effect in the pollutant-exposed groups compared to sham control animals and to assess the type, and extent, of any toxicological interaction between acid and ozone. Interaction was considered to occur when the effects of combined exposure were either significantly greater or less than additive. Pollutant exposures had no effect on lavage fluid levels of lactate dehydrogenase, prostaglandins E2 and F2 alpha, nor on the numbers, viability, or types of immune cells recovered by lavage. Phagocytic activity of macrophages was depressed at the two highest acid levels and at all levels of ozone. Exposure to all mixtures showed significant antagonism. Superoxide production by stimulated macrophages was depressed by acid exposure at the two highest concentrations, while ozone alone had no effect. Significant antagonistic interaction was observed following exposure to mixtures of 75 or 125 micrograms/m3 acid with 0.1 or 0.3 ppm ozone. The activity of tumor necrosis factor elicited from stimulated macrophages was depressed by acid at 75 and 125 micrograms/m3 while ozone had no effect. Exposure to mixtures of 125 micrograms/m3 acid with 0.3 or 0.6 ppm ozone resulted in synergistic interaction. This study provided additional evidence for antagonism between two common air pollutants and demonstrated that the type of interaction between sulfuric acid and ozone depended upon the endpoint but that the magnitude of any interaction was not always related to the exposure concentrations of the constituent pollutants

Previous work from this laboratory demonstrated that neonatal rats and postweanling rabbits are more sensitive to ozone-induced stimulation of pulmonary arachidonic acid (AA) metabolism than are young adults (Fundam. Appl. Toxicol. 15, 779.) In the study reported here, we have extended our initial investigation to include the influence of animal age on temporal aspects of pulmonary AA metabolism and several other responses to brief exposures to 1 ppm ozone. Rats of discrete ages ranging from 13 days to 16 weeks were exposed to 1 ppm ozone or to air for 2, 4, or 6 hr. Immediately following exposure the lungs were lavaged with six consecutive volumes of phosphate-buffered saline and the acellular fluid from the first lavage volume recovered was analyzed for its content of prostaglandin E2 (PGE2), protein, and lactate dehydrogenase. Leukocytes recovered by lavage were quantitated and characterized by viability and percentage of polymorphonuclear (PMN) cells. Several lines of evidence verified that PGE2 was produced by the lung as a consequence of ozone exposure and that its concentration in the fluid from the first lavage was a reasonably good index of pulmonary AA metabolism to prostanoids. We also demonstrated that the lavage process itself stimulates the lung, resulting in increased AA metabolism to prostanoids that were recovered in the second and following lavage volumes. The time course of PGE2 production by the ozone-exposed lung varied considerably with animal age. Neonatal rats 13 days of age were the most sensitive to ozone stimulation. At 2 hr of exposure, PGE2 concentration in the first lung lavage of these animals peaked at values approximately two orders of magnitude above controls and then decreased sharply with continued exposure. Adults and older neonates (18 days of age) were much less responsive to 2-hr exposures; however, continued exposure of these rats for up to 6 hr resulted in increasing PGE2 concentration in the first lung lavage. Other responses showed various degree of age dependence. The percentage of lavaged leukocytes that were nonviable (i.e., trypan blue-positive) showed a strong inverse correlation with animal age. In 13-day-old rats that were exposed for 6 hr, the percentage of dead leukocytes reached nearly 50%. In addition, sheets or clumps of dead cells that were judged to be epithelial cells were lavaged from these animals. Conversely, 16-week-old adult males exposed to ozone for 6 hr showed little evidence of damage to cells of the respiratory tract.(ABSTRACT TRUNCATED AT 400 WORDS)

Although several epidemiological studies have provided evidence that airborne sulfate particles can produce adverse health effects in susceptible individuals, there is only limited data demonstrating respiratory effects in human volunteers and experimental animals at near ambient concentrations. We have demonstrated previously that the mixing of metal oxide particles with SO2 under humid conditions produces acid-coated particles that are significantly more potent in causing pulmonary function changes than pure acid droplets. The present study examined the nonspecific airway responsiveness to acetylcholine in guinea pigs exposed to acid-coated zinc oxide particles. One and a half hours after a 1-h exposure to the aerosols or a control atmosphere, pulmonary resistance (RL) was measured in awake, spontaneously breathing animals before and during a challenge with increasing doses of iv acetylcholine (Ach). The provocative infusion rate of Ach that resulted in a 100% increase in RL (PR100) was significantly decreased (p less than .05) in animals exposed to sulfuric acid-coated metal oxide particles (approximately 30 micrograms/m3 sulfate) compared to control animals exposed to furnace gases (79.6 +/- 19.4 vs. 179.6 +/- 16.2 micrograms/kg/min, mean +/- SE, respectively). The PR100 of animals exposed to SO2 (109.1 +/- 45.4) or metal oxide particles (106.7 +/- 38.1) alone was not significantly different from that of furnace gas control animals, indicating that the acid coating on the metal oxide particles and not the particles themselves or the SO2 was responsible for the decrease in the PR100. Moreover, a 10-fold greater amount of total sulfate as a pure aqueous sulfuric acid aerosol was necessary to produce a decrease in PR100 (88.6 +/- 11.0 micrograms/kg/min) equivalent to that produced by coated particles. These results suggest that acute exposure to near-ambient concentrations of sulfuric acid under conditions that promote the formation of acid as a surface coating in respirable particles can induce a nonspecific airway hyperresponsiveness. In a similar manner, a dose-dependent significant decrease in PR100 was also produced in animals exposed to sodium sulfite droplets. Thus a single exposure to different forms of sulfur oxide aerosols can induce an alteration in the responsiveness of airway smooth muscle in the guinea pig

Acidic sulfate is the most toxicologically important sulfur oxide which exists in the ambient air. To determine if particle size influences toxic effects of sulfuric acid, we investigated the effects of sulfuric acid aerosols of two different sizes on biochemical and cellular parameters of bronchoalveolar lavage fluid from exposed guinea pigs. Guinea pigs were exposed to fine (mass median diameter, 0.3 micron), and ultrafine (mass median diameter, 0.04 micron) sulfuric acid aerosols at 300 micrograms/m3 for 3 hr/day. The animals were euthanized immediately and 24 hr after 1 and 4 days of exposure and lungs were lavaged. Elevated beta-glucuronidase, lactate dehydrogenase activities, and total protein concentration as well as decreased cell viability were observed in the lavage after a single exposure to sulfuric acid aerosols of both sizes. These alterations were small, though statistically significant, and transient. No alteration in these parameters was observed after 4 days of exposure to acid aerosols. In contrast, sulfuric acid-induced alterations in alveolar macrophage function were more pronounced and longer lasting. Immediately after a single exposure to fine acid, there was a 2.7-fold increase in the spontaneous tumor necrosis factor (TNF) release over that in the control group while endotoxin-stimulated TNF release was increased by 2.2-fold. In addition, acid aerosols of both sizes increased the TNF release from macrophages after 4 days of exposure, although there was no clear temporal pattern of induction or recovery. Furthermore, immediately after 4 days of exposure to either fine or ultrafine acid, the amount of H2O2 that could be induced from baseline production by alveolar macrophages was 2.2-fold higher than that of the controls. The phagocytic function of macrophages was also altered by exposure to sulfuric acid aerosols. Twenty-four hours after single or multiple exposure, fine acid enhanced (as high as 78% above control) the in vitro phagocytic activity of alveolar macrophages while ultrafine acid depressed the phagocytic capacity (as much as 50% below that in the control). In addition to these biochemical parameters and cellular functions, we also measured the intracellular pH (pHi) of macrophages harvested after exposures to these acid aerosols using a pH-sensitive fluorescent dye. The resting pHi was depressed after a single exposure to both acid aerosols. The depression in pHi persisted 24 hr after ultrafine acid exposure.(ABSTRACT TRUNCATED AT 400 WORDS)

Urban air pollution in the United States is composed of a complex mixture of particles and gases. Among the most prominent products of the atmospheric pollutants are sulfur oxides and ozone. In this report, we use two exposure protocols to examine the interaction between exposure to these two pollutants. In the first exposure regimen, guinea pigs were exposed to sulfuric acid (pure sulfuric acid mist or sulfuric acid layered on ZnO) for 1 h. Each exposure is followed 2 h later by another exposure to 0.15 ppm ozone for 1 h. Pulmonary function parameters were measured immediately after the ozone exposure. In guinea pigs that were exposed to 300 micrograms/m3 pure sulfuric acid mist, subsequent exposure to 0.15 ppm ozone did not produce additional change in pulmonary functions. In guinea pigs that were exposed to 84 micrograms/m3 sulfuric acid layered on ZnO, subsequent exposure to 0.15 ppm ozone produced more than additive alterations in vital capacity and diffusing capacity. In the second exposure regimen, guinea pigs were exposed to 24 micrograms/m3 sulfuric acid layered on ZnO for 3 h/d for 5 d. On d 8 and 9, animals received two additional daily 3-h exposures to 24 micrograms/m3 sulfuric acid layered on ZnO, and pulmonary functions were measured at the end of the daily exposure. Greater reductions in lung volumes and diffusing capacity were observed in animals on d 9 than would be observed in animals that received no additional exposure. In the third exposure regimen, guinea pigs were exposed to 24 micrograms/m3 sulfuric acid layered on ZnO for 3 h/d for 5 d. On d 9, animals were exposed to 0.15 ppm ozone for 1 h and pulmonary functions were measured at the end of the ozone exposure. Ozone exposure on d 9 induced reductions in lung volumes and diffusing capacity that were not observed in animals receiving exposures to either ozone or sulfuric acid layered ZnO alone. We conclude that single or multiple exposure to sulfuric acid-layered ZnO sensitizes guinea pigs to subsequent sulfuric acid or ozone exposure

Spontaneous hippocampal EEG activity and evoked field potentials were investigated in intact rats and in animals with fetal hippocampal grafts. Pieces of hippocampal grafts, derived from 15- to 16-day-old fetuses, were used to prepare cell suspensions and grafted directly into the intact hippocampus. Control animals received suspension grafts of the cerebellum derived from fetuses of identical age. Host hippocampal electrical patterns were monitored with chronic single electrodes or with a 16-microelectrode probe from 7 to 10 months after grafting. In contrast to previously reported high survival rates of fetal grafts in studies with damage to the host brain prior to grafting, survival of both hippocampal (60%) and cerebellar grafts (20%) was very poor in the intact hippocampus. In animals with cerebellar transplants or without surviving grafted neurons the electrical activity of the host hippocampus was indistinguishable from normal controls. In rats with hippocampal grafts short duration, large amplitude EEG spikes (up to 10 mV) were recorded, predominantly during immobility. When the EEG spikes (putative interictal spikes) were of large amplitude and contained population spikes, test evoked responses delivered to the perforant path were suppressed after the spontaneous events. In contrast, evoked responses were facilitated by interictal spikes without population spikes. The threshold of electrically induced afterdischarges did not differ significantly between groups of intact rats and animals with or without hippocampal grafts. However, in three rats with hippocampal grafts the evoked afterdischarges were associated with behavioral seizures. In two of these rats spontaneously occurring seizures were also observed. Synaptophysin-immunoreactivity demonstrated growth of the host mossy fibers into the graft.(ABSTRACT TRUNCATED AT 250 WORDS)

The authors have developed a system that generates copper oxide aerosol similar to the primary emissions from smelters. The surface of the ultrafine copper oxide aerosol is coated with a layer of sulfur oxides consisting of sulfate, S(VI), and sulfite, S(IV). Guinea pigs were exposed to this sulfur oxide layered copper oxide aerosol, and pulmonary mechanical functions were measured by using the Amdur-Mead method. The concentration of sulfur oxides on the aerosol was determined by using a flame photometric detector system. Although sulfuric acid was not found in this system, S(IV) at concentrations as low as 0.36 mumol/m3 delivered as a surface layer caused prolonged changes in pulmonary mechanical functions