Inhaled particle retention in rats receiving low exposures of diesel exhaust.

To study the effects of a low concentration exposure on the retention and clearance of submicron particles from the lungs, we exposed male Fisher 344 rats to diesel exhaust diluted to 50 micrograms diesel exhaust particles (DP)/m3, 20 h/d, 7 d/wk for 52 wk. Lung burdens (amount of DP in lungs) and the alveolar macrophage burdens were measured up to 52 wk postexposure. By 1 yr postexposure at least 80% of the DP was eliminated from the lungs and similarly cleared from the lavaged pool of macrophages. The DP remaining in the lungs was observed in alveolar, parabronchial and paravascular maculae. In contrast to previous high concentration exposure studies, only trace amounts of particles were observed in the mediastinal lymph nodes. To study the concentration dependence of particle retention, rats were exposed to equivalent exposures of 18 d x mg DP/m3 delivered at 5700 micrograms/m3 for 3 d, 1600 micrograms/m3 for 12 d, 250 micrograms/m3 for 72 d, or 50 micrograms/m3 for 365 d. Higher lung and macrophage burdens were initially achieved with the brief, high concentration exposures. During the postexposure period, animals exposed to the higher concentrations cleared more of the lung burden. Exposure to lower concentrations resulted in higher long-term lung burdens. These results are consistent with a model of lung clearance in which the macrophage burden and the duration of exposure are both important to the formation of the maculae. In a brief high concentration exposure, the macrophage burden rises rapidly, but then declines rapidly. However, in longer low concentration exposures, the macrophage burden will not reach the same peak, but stays at intermediate levels during the exposure and stimulates a steady development of the lung maculae from particle-laden macrophages leaving the active pool of pulmonary phagocytes.     

Ultrastructure and morphometry of the alveolar lung of guinea pigs chronically exposed to diesel engine exhaust: six month's experience

The impact of chronic inhalation of diesel exhaust (DE) on alveolar lung was studied in 24 Hartley guinea pigs. Groups were sacrificed sequentially at 2 weeks, 3 months and 6 months of exposure to either 750 micrograms or 1500 micrograms DE particles (DEP) per m3 along with age-matched concurrent controls. Although qualitative ultrastructural changes were noticed during this time interval and dosage schedule, there was no evidence of pathologic changes such as fibrosis or emphysema. The cellular uptake of DEP was striking. By 2 weeks three alveolar cell types (alveolar macrophages, epithelial type 1 cells and interstitial macrophages) plus one type of granulocytic leukocyte (eosinophils) confined DEP within phagosomes without evidence of cytotoxicity. A certain phagosomal DEP population had a bull's eye appearance and diameters of 0.0727 +/- 0.01 micron. Morphometric analysis applied to electron micrographs demonstrated that arithmetic mean tissue thickness of the air-blood barrier was generally increased (p less than 0.05) during DE exposure. For the 750 micrograms DE sets, the increase over control (1.56 micron) was 41% at 2 weeks, 46% at 3 months and 77% at 6 months while the 1500 micrograms DE set at 6 months exceeded control by 130%. Increases in absolute tissue volumes of interstitium and epithelial type 2 cells largely accounted for the increased tissue thickness. Harmonic mean tissue thickness for controls remained near 0.537 +/- 0.03 micron for the study interval, contrasting with values for 3 and 6 months 750 micrograms DE and 6 months 1500 micrograms DE sets which increased. However, the diffusion capacity of the lung determined morphometrically was not decreased in DE exposed sets. Although cellular uptake of DEP and increased prominence of secretory epithelium were dose/duration related, absence of linearity suggests adaptative responses.     

Aryl hydrocarbon hydroxylase induction in rat pulmonary alveolar macrophages (PAMs) by diesel particulates and their extracts

The Aryl hydrocarbon hydroxylase (AHH) activity in the lavaged PAMs was investigated in male Fischer rats after inhalation of diluted diesel exhaust (DE), and compared with intratracheal instillation of the organic solvent extract of hydrocarbons adsorbed on the particulate surface. Animals were exposed to concentrations of 1500 micrograms/m3 or 6000 micrograms/m3 of diesel particulates, 20 hrs/day, 7 days/wk for 2 days up to 28 days, or treated with a single intratracheal dose of DE extract or benzo[a]pyrene (B[a]P) dissolved in gelatin-saline solution. The counts of lavageable PAM and polymorphonuclear leukocytes (PMN) were significantly elevated in the exposed rats. However, a decrease of AHH activity was found in the PAM cells after four weeks of exposure to both particulate concentrations. The AHH activity in the PAMs from rats injected intratracheally with 5 mg/kg of B[a]P was slightly increased; no change was observed in PAMs similarly treated with DE extract. Enzyme induction is not noted because of absence of sufficient quantities of hydrocarbons need for induction. This could be further potentiated that macrophages metabolize and perhaps reduce hydrocarbon levels by translocation.     

Aryl hydrocarbon hydroxylase activity induced by injected diesel particulate extract vs inhalation of diluted diesel exhaust

The effects of long-term inhalation of diluted diesel exhaust on aryl hydrocarbon hydroxylase activity (AHH) and cytochrome P-450 content in lung and liver microsomes were investigated in male Fischer-344 rats and compared with repeated parenteral administration of organic solvent extracts of hydrocarbons adsorbed on the diesel particulate surface during the combustion process. The animals were exposed to concentrations of 750 micrograms m-3 or 1500 micrograms m-3 of diesel particulates from a 5.7L GM diesel engine 20 h per day, 5 1/2 days per week for up to 9 months or treated by repeated IP injections of diesel particulate extract (dissolved in corn oil) from the same engine at several dose levels for 4 days. No significant effects of long-term inhalation exposure were observed in liver microsomal AHH activity. A slight decrease in lung microsomal AHH activity was found in rats following 6 months of exposure to diesel exhaust at the particulate concentration of 1500 micrograms m-3. The total mass of particles deposited in the lung during the inhalation exposure was estimated and an equivalent dose of extractable hydrocarbons was administered intraperitoneally; no increase in AHH activity was observed in the lung or liver microsomes. In contrast, 1.4- to 9-fold increases in AHH activity were observed in liver and lung microsomes of rats pretreated by intraperitoneal doses 10-50 times larger than the most conservative estimate of the deposited lung burden. No changes in cytochrome P-450 content were observed in the microsomes of rat liver after inhalation or injection treatment studies.(ABSTRACT TRUNCATED AT 250 WORDS)     

Experimental conditions in GMR chronic inhalation studies of diesel exhaust

A chronic inhalation exposure study was initiated to study the potential health effects of diesel exhaust on laboratory animals. Test atmospheres of clean air (control) or freshly diluted diesel exhaust at nominal particulate concentrations of 250, 750 and 1500 micrograms m-3 were supplied to four large volume inhalation chambers in which individually housed Fischer 344 albino rats (Rattus norvegicus) and Hartley guinea pigs (Cavia porcellus) were exposed for 20 h per day, 51/2 days per week. The diesel aerosol concentration, chamber temperature and relative humidity were continually monitored and controlled to maintain the exposure dose levels and an environment of 22 +/- 2 degrees C and 50 +/- 20% relative humidity. Animals were randomly sampled from the chambers for physiological, biochemical and pathological studies throughout the exposure period. The study was continued without interruption for 24 months with the mean diesel particle mass concentrations within 6% of the target values. The standard deviation of the mass concentration measurements was approximately 30% of the mean.     

A spectrophotometric method for the quantitation of diesel exhaust particles in guinea pig lung

Particulate material in diesel engine exhaust deposits in the lungs of exposed animals. A technique for measuring the amount of soot in the lungs would be useful for determining the rates of the deposition and clearance of the submicron particles. The paper describes the use of light extinction for quantitating diesel particles in aqueous suspension. Particles collected by electrostatic precipitation and finely suspended in 0.01 N NaOH by sonication strongly absorb visible light. The extinction of light at 750 nm is proportional to the mass concentration of particles, with an extinction coefficient of 28 +/- 1 cm2 mg-1. Lungs from guinea pigs exposed to dilute diesel exhaust were dried and digested in potassium hydroxide and ethanol. The insoluble particles were centrifuged and resuspended in water by sonication. The optical density of the suspension was compared to that of suspensions made from lungs of animals not exposed to diesel exhaust, with or without known amounts of particulate added at the beginning of digestion. A concentration-dependent increase in the total amount of particles per lung was found for guinea pigs exposed to 0, 269, 813 and 1530 micrograms m-3 diesel particles for 6 months.     

Effect of exposure to diesel exhaust on pulmonary prostaglandin dehydrogenase (PGDH) activity

There has been evidence that an acute exposure of laboratory animals to nitrogen dioxide (NO2) for a short period of time can cause marked inhibition of pulmonary PGDH activity. Since diesel exhaust contains NO2, this investigation was undertaken to determine the effect of long-term exposure of guinea pigs and rats to diesel exhaust. The study involves measurement of PGDH activity in the lung tissue as obtained from these animals following exposure to 250 and 1500 micrograms m-3 of diesel particles for various time periods in relation to the appropriate time-matched controls. In guinea pigs, exposure for 6 weeks to 250 micrograms m-3 of diesel exhaust seems to stimulate the PGDH activity about two-fold in comparison to the time-matched controls while, paradoxically, the exposure to 1500 micrograms m-3 of diesel exhaust did not show any particular effect. The exposures to diesel exhaust for 12 weeks, as well as 24 weeks, seem to show concentration dependent lowering of PGDH activity as compared to the time-matched controls. This study also documents the well-known species difference in PGDH activity in that the rat shows much lower activity of this enzyme than the guinea pig. Because of undetectable enzyme activity in many samples, it has not been possible to draw any meaningful conclusion as to the effect of diesel exposure on the enzyme in rat.     

Characteristics of alveolar macrophages following the deposition of a low burden or iron oxide in the lung

Rats were exposed to aerosols of iron-59 oxide (mass median aerodynamic diameter, MMAD = 1.6 micron, sigma g = 3.0) at a nominal concentration of 20 mg/m3 for 2 h to determine how a low lung burden (approximately 30 micrograms) of innocuous particles affects the size of the alveolar macrophage (AM) pool, and the functional status of the AM as assessed in vitro by their ability to exclude Trypan blue, adhere to plastic substrate, and bind and phagocytize sheep erythrocytes opsonized with immunoglobulin G (SRBC-IgG). Iron oxide deposition did not bring about significant changes in cell types or numbers of AM lavaged, AM viabilities, or the plastic substrate adherence characteristics of the AM. As of 1 d post exposure, however, the ability of AM to phagocytize SRBC-IgG increased. Phagocytosis was maximally enhanced 3-7 d post exposure and returned to control levels by 20 d after exposure. The increase in phagocytic activity correlated with an increase in AM avidities for SRBC-IgG. The kinetics of subsidence of the phagocytic response did not parallel the alveolar clearance rate of the deposited particles [t1/2 (biol)/53 d]. These studies show the deposition of a low lung burden of a noncytotoxic dust can transiently enhance Fc gamma-receptor-mediated particle binding and phagocytosis by AM.     

Pulmonary function testing of animals chronically exposed to diluted diesel exhaust

The purpose of this work was to assess the potential effect that chronic inhalation of diesel exhaust may have on lung mechanics and lung volume. Noninvasive pulmonary function tests that produced data on lung air flows and volumes have been conducted repeatedly on 25 male Fischer-344 rats exposed to diesel exhaust at a particulate concentration of 1500 micrograms m-3, 20 h per day, 5 1/2 days per week, for 612 days. The same tests were conducted on 25 clean air control animals. When the data were normalized, the majority of tests did not reveal any significant deviation from the norm for the first year of exposure. In the second year, the functional residual capacity and its component volumes - expiratory reserve and residual volume, maximum expiratory flow at 40% of vital capacity, maximum expiratory flow at 20% of vital capacity and the forced expiratory volume in 0.1 s - were significantly greater in the diesel exposed animals. The data are inconsistent with known clinically significant adverse health effects. Although the lung volume changes in the diesel exposed animals could be indicative of emphysema or other forms of chronic obstructive lung disease, this interpretation is contradicted by the air flow data which suggest simultaneous lowering of the resistance of the smaller airways. The observations are not consistent with documented clinical lung disease in man.     

Metabolism of 1-[14C]nitropyrene in respiratory tract tissue of rats exposed to diesel exhaust

The purpose of this study was to determine how prior exposure of rats to graded concentrations of diesel exhaust would affect respiratory tract tissue metabolism of 1-nitropyrene (NP), a known constituent of diesel exhaust. Rats were exposed (whole body) 7 hr/day, 5 days/week for 4 weeks to clean air (controls) or to diluted diesel exhaust containing concentrations of 350, 3300, and 7400 micrograms particles/m3. After exposures, nasal tissue and lungs from rats were tested for their ability to metabolize NP. Rat nasal tissue was incubated for 10 min with 20 microM 1-[14C]NP. Isolated perfused rat lungs were perfused for 90 min with 25 microM 1-[14C]NP. NP metabolites formed in rat nasal tissue and the isolated perfused rat lung were separated by high-pressure liquid chromatography. Exposure of rats to 7400 micrograms particles/m3 for 4 weeks resulted in significant increases (twofold) in rates of NP metabolism in both nasal tissue (440 pmol/mg protein/min) and the isolated perfused rat lung (112 nmol/g lung). Exposure of rats to lower concentrations of diesel exhaust did not increase the rates of NP metabolism in either rat nasal tissue or perfused rat lungs. In all cases, the major metabolites of NP formed in nasal tissue and perfused lungs were 3-, 6-, and 8-hydroxy-1-nitropyrene and 4,5-dihydro-4,5-dihydroxy-1-nitropyrene. A fourfold increase was observed in the amounts of 14C covalently bound in lungs from rats exposed to 7400 micrograms particles/m3. The results from this study indicate that exposure to diesel exhaust particles significantly alters metabolism and subsequent covalent binding of NP.     

Species differences in release of arachidonate metabolites in response to inhaled diluted diesel exhaust

In life-span studies in CD-1 mice and F344/Crl rats, inhaled diluted diesel exhaust was highly fibrogenic in rats but not in mice. This was the case despite the higher lung burden, in mg soot/g lung, achieved in mice compared to rats. We tested the hypothesis that the greater fibrogenicity of the soot in rats was due in part to greater release of mediators of inflammation from alveolar cells in rats compared to mice. Female F344/rats and B6C3F1 mice were exposed for up to 17 days to diluted diesel exhaust containing 3.5 mg/m3 of soot. The lungs of control and soot-exposed animals were lavaged after 2, 12 or 17 days of exposure. The presence of leukotriene (LT)B4, LTC4, prostaglandin (PG)E2, PGF2 alpha and thromboxane (TX) B2 in the lavage fluids and LTB4 and PGF2 alpha in cultured lavage cell supernatants was determined. The total amount of each lavage fluid constituent was normalized to lung weight for species comparisons. Control rats had higher levels of TXB2 (16-fold), and LTB4 (6-fold) and PGE2 (2-fold) than control mice, but control mice had higher amounts of LTC4 (4-fold). Control rats and mice had approximately the same amounts of PGF2 alpha/g lung in bronchoalveolar lavage fluid (BALF). Rats exposed to diesel exhaust had increases in BALF PGF2 alpha and LTB4 that were highest after 2 days of exposure and decreased thereafter. Mice had lesser increases in both parameters. Rat cells recovered from lavage fluid released larger amounts of LTB4 into culture supernatants than mouse cells. The data were consistent with the hypothesis that soot-laden rat alveolar cells release greater quantities of mediators of inflammation than do the alveolar cells in mice.     

DOSE-RESPONSE ASSESSMENT AND EFFECT OF PARTICLES IN GUINEA PIGS EXPOSED CHRONICALLY TO DIESEL EXHAUST: ANALYSIS OF VARIOUS BIOLOGICAL MARKERS IN PULMONARY ALVEOLAR LAVAGE FLUID AND CIRCULATING BLOOD

To elucidate dose-response and other effects of diesel particles in guinea pigs chronically exposed to diesel exhaust, various biomarkers for chronic obstructive lung diseases were studied using bronchoalveolar lavage (BAL) fluid and blood specimens. Guinea pigs were exposed 16 h/day, 6 days/wk, for 6, 12, 18, or 24 mo to filtered air (control group, n = 8-10), a low level of diesel exhaust (L group: NO 2 = 0.22 ± 0.03 ppm; SO 2 = 0.6 ± 0.19 ppm; particles = 0.21 ± 0.07 mg/m 3, n = 8-10), medium level of diesel exhaust (M group; NO 2 = 1.07 ± 0.09 ppm; SO 2 = 2.83 ± 0.73 ppm; particles = 1.14 ± 0.26 mg/m 3, n = 8-10), and high level of diesel exhaust (H group: NO 2 = 2.88 ± 0.29 ppm; SO 2 = 6.49 ± 1.75 ppm; particles = 2.94 ± 0.69 mg/m 3, n = 8-10), or at a medium concentration of diesel exhaust without particulate matters (MG group: NO 2 = 1.01 ± 0.09 ppm; SO 2 = 2.66 ± 0.64 ppm; particles = 0.01 ± 0.01 mg/m 3, n = 8-10). Anesthetized animals were sacrificed and BAL fluid from the lung and blood from right ventricle were collected. Various biomarkers of inflammation, components of mucus and surfactant, bronchoconstrictors were determined. Changes of leukotriene C4 in plasma, eosinophil counts, biomarkers of inflammation and cytotoxicity, and mucus and surfactant components in BAL fluid were statistically different among the C, L, M, and H groups after adjustment for the exposure period and group-by-exposure period with respect to their interactions in two-way analysis of variance (ANOVA). The levels of these biomarkers in the H group were higher than those of the M group, whereas those of the L group showed no significant changes compared with those of the C group during experimental period. Onset of significant changes of these biomarkers for the M group was at 18 mo of exposure, whereas that for the H group was at 12 mo of exposure, which resulted in changes in the levels of biomarkers in BAL fluid. Although numbers of eosinophils in BAL fluid increased significantly in the M and H groups at 12 mo, only leukotrine C4 increased at 18 and 24 mo in blood and at 24 mo in BAL fluid. Animals exposed to the medium level of diesel exhaust without particulate matter showed significantly less increase of these biomarkers as compared with animals exposed to the same level of diesel exhaust with particulate matters. These findings indicate that chronic exposure to diesel exhaust induced continuous inflammation, overproduction of mucus, and phospholipids in the lung. Animals exposed to the high dose of diesel exhaust showed a plateau of biological responses at 12 mo of exposure. Particulate matter in diesel exhaust appears to play an important role in development of lung injury by chronic emission exhaust exposure.     

Dose-response assessment and effect of particles in guinea pigs exposed chronically to diesel exhaust: analysis of various biological markers in pulmonary alveolar lavage fluid and circulating blood

To elucidate dose-response and other effects of diesel particles in guinea pigs chronically exposed to diesel exhaust, various biomarkers for chronic obstructive lung diseases were studied using bronchoalveolar lavage (BAL) fluid and blood specimens. Guinea pigs were exposed 16 h/day, 6 days/wk, for 6, 12, 18, or 24 mo to filtered air (control group, n = 8-10), a low level of diesel exhaust (L group: NO(2) = 0.22 +/- 0.03 ppm; SO(2) = 0.6 +/- 0.19 ppm; particles = 0.21 +/- 0.07 mg/m(3), n = 8-10), medium level of diesel exhaust (M group; NO(2) = 1.07 +/- 0.09 ppm; SO(2) = 2.83 +/- 0.73 ppm; particles = 1.14 +/- 0.26 mg/m(3), n = 8-10), and high level of diesel exhaust (H group: NO(2) = 2.88 +/- 0.29 ppm; SO(2) = 6.49 +/- 1.75 ppm; particles = 2.94 +/- 0.69 mg/m(3), n = 8-10), or at a medium concentration of diesel exhaust without particulate matters (MG group: NO(2) = 1.01 +/- 0.09 ppm;#10; SO(2) = 2.66 +/- 0.64 ppm; particles = 0.01 +/- 0.01 mg/m(3), n = 8-10). Anesthetized animals were sacrificed and BAL fluid from the lung and blood from right ventricle were collected. Various biomarkers of inflammation, components of mucus and surfactant, bronchoconstrictors were determined. Changes of leukotriene C4 in plasma, eosinophil counts, biomarkers of inflammation and cytotoxicity, and mucus and surfactant components in BAL fluid were statistically different among the C, L, M, and H groups after adjustment for the exposure period and group-by-exposure period with respect to their interactions in two-way analysis of variance (ANOVA). The levels of these biomarkers in the H group were higher than those of the M group, whereas those of the L group showed no significant changes compared with those of the C group during experimental period. Onset of significant changes of these biomarkers for the M group was at 18 mo of exposure, whereas that for the H group was at 12 mo of exposure, which resulted in changes in the levels of biomarkers in BAL fluid. Although numbers of eosinophils in BAL fluid increased significantly in the M and H groups at 12 mo, only leukotriene C4 increased at 18 and 24 mo in blood and at 24 mo in BAL fluid. Animals exposed to the medium level of diesel exhaust without particulate matter showed significantly less increase of these biomarkers as compared with animals exposed to the same level of diesel exhaust with particulate matters. These findings indicate that chronic exposure to diesel exhaust induced continuous inflammation, overproduction of mucus, and phospholipids in the lung. Animals exposed to the high dose of diesel exhaust showed a plateau of biological responses at 12 mo of exposure. Particulate matter in diesel exhaust appears to play an important role in development of lung injury by chronic emission exhaust exposure.     

Chronic diesel exhaust exposures of rats demonstrate concentration and time-dependent effects on pulmonary inflammation

Long-term, repeated exposure to particles in air pollution increases the risk for chronic respiratory diseases and cardiorespiratory mortality. The biological linkages remain poorly understood in chronic exposure to particle matter. To elucidate and verify these linkages, we investigated long-term exposure to diesel emission with respect to dose dependence and the effect of components without particles from diesel emission in rats. Wistar rats were exposed to filtered air (C group), diesel exhaust at low (L group), medium (M group), and high level (H group), or at a medium concentration diesel exhaust without particulate matter (MG group), for 16 h/day, 6 days/wk, for 6, 12, 18, or 24 mo. Anesthetized animals were sacrificed and bronchoalveolar lavage (BAL) fluid from the lung and blood from the right ventricle were collected. Various biomarkers of inflammation and components of mucus and surfactant were determined. Changes in total cell counts and cell differentiation, total protein, mucus and surfactant components, and prostaglandin E(2) in BAL fluid, but not biomarkers in plasma, showed statistical differences among the C, L, M, and H groups during the experimental period. The changes in these biomarkers in the H group were greater than those in the M group, whereas those in the L group showed no significant changes compared with those in the C group during the experimental period. The onset of significant changes in inflammatory cells and these biomarkers in BAL fluid for the M and H groups was at 6 to 12 mo of exposure. The maximum level was reached at 12 to 18 mo of exposure. Although BAL prostaglandin E(2) decreased significantly at 6 mo of exposure in the M and H groups, this trend was not observed in the C and L groups. Animals exposed to a medium level of diesel exhaust without particulate matter showed significantly less inflammatory cells and various biomarkers in BAL fluid than animals exposed to the same level of diesel exhaust with particulate matter during the experimental period. These findings suggest that biological response to inhaled particles is aggravated during chronic exposure to diesel exhaust dose-dependently. Inflammation and overproduction of mucus and surfactant components reached a plateau at 12 or 18 mo of exposure during a 24-mo experimental period. No adverse effect of particles (less than 1.0 mg particles/m(3) of diesel emission) was observed in these rats. However, our data suggest that particulate matter plays an important role during development of chronic lung injury induced by diesel emission exhaust.     

Tumours of the respiratory tract in rats and hamsters following chronic inhalation of engine exhaust emissions

The potential carcinogenic effect of inhaled automobile exhaust emissions was examined in rodents. Both rats and hamsters were exposed to the emissions from (1) a gasoline engine, (2) a gasoline engine fitted with a three-way catalytic converter, (3) a diesel engine and (4) a diesel engine with particle filtration. Exposures were for 16 hours per day, 5 days per week, for 2 years. All hamsters were sacrificed at the end of the 2-year exposure period, whereas the rats surviving after 2 years of exposure were maintained for a further 6-month observation period without additional exposure to emissions. Some of the hamsters in each treatment group were pretreated with diethylnitrosamine to induce respiratory tract tumours. No statistically significant changes were seen in the incidence of respiratory tract tumours in emission-exposed hamsters compared to controls. This lack of a treatment-related effect was seen in both the nitrosamine pretreated and the non-pretreated hamsters. There was no increase in the incidence of lung tumours in rats exposed to filtered diesel exhaust or to the exhaust from the gasoline or gasoline-catalyst engines. There was a statistically significant increase in the incidence of lung tumours in rats exposed to diesel engine emissions compared to controls. A clear dose response was evident in both males and females, although the incidence of lung tumours was markedly higher in females (96% in rats surviving beyond 2 years) than in males (44% in rats surviving beyond 2 years). An increased incidence of lung tumours was observed only in rats exposed to mean concentrations of diesel soot particles of either 2200 or 6600 micrograms/m3.(ABSTRACT TRUNCATED AT 250 WORDS)     

Chronic Diesel Exhaust Exposures of Rats Demonstrate Concentration and Time-Dependent Effects on Pulmonary Inflammation

Long-term, repeated exposure to particles in air pollution increases the risk for chronic respiratory diseases and cardiorespiratory mortality. The biological linkages remain poorly understood in chronic exposure to particle matter. To elucidate and verify these linkages, we investigated long-term exposure to diesel emission with respect to dose dependence and the effect of components without particles from diesel emission in rats. Wistar rats were exposed to filtered air (C group), diesel exhaust at low (L group), medium (M group), and high level (H group), or at a medium concentration diesel exhaust without particulate matter (MG group), for 16 h/day, 6 days/wk, for 6, 12, 18, or 24 mo. Anesthetized animals were sacrificed and bronchoalveolar lavage (BAL) fluid from the lung and blood from the right ventricle were collected. Various biomarkers of inflammation and components of mucus and surfactant were determined. Changes in total cell counts and cell differentiation, total protein, mucus and surfactant components, and prostaglandin E 2 in BAL fluid, but not biomarkers in plasma, showed statistical differences among the C, L, M, and H groups during the experimental period. The changes in these biomarkers in the H group were greater than those in the M group, whereas those in the L group showed no significant changes compared with those in the C group during the experimental period. The onset of significant changes in inflammatory cells and these biomarkers in BAL fluid for the M and H groups was at 6 to 12 mo of exposure. The maximum level was reached at 12 to 18 mo of exposure. Although BAL prostaglandin E 2 decreased significantly at 6 mo of exposure in the M and H groups, this trend was not observed in the C and L groups. Animals exposed to a medium level of diesel exhaust without particulate matter showed significantly less inflammatory cells and various biomarkers in BAL fluid than animals exposed to the same level of diesel exhaust with particulate matter during the experimental period. These findings suggest that biological response to inhaled particles is aggravated during chronic exposure to diesel exhaust dose-dependently. Inflammation and overproduction of mucus and surfactant components reached a plateau at 12 or 18 mo of exposure during a 24-mo experimental period. No adverse effect of particles (less than 1.0 mg particles/m 3 of diesel emission) was observed in these rats. However, our data suggest that particulate matter plays an important role during development of chronic lung injury induced by diesel emission exhaust.     

Nonparticulate components of diesel exhaust promote constriction in coronary arteries from ApoE-/- mice.

Air pollution is positively associated with increased daily incidence of myocardial infarction and cardiovascular mortality. We hypothesize that air pollutants, primarily vapor phase organic compounds, cause an enhancement of coronary vascular constriction. Such events may predispose susceptible individuals to anginal symptoms and/or exacerbation of infarction. To develop this hypothesis, we studied the effects of nonparticulate diesel exhaust constituents on (1) electrocardiographic traces from ApoE-/- mice exposed whole-body and (2) isolated, pressurized septal coronary arteries from ApoE-/- mice. ApoE-/- mice were implanted with radiotelemetry devices to assess electrocardiogram (ECG) waveforms continuously throughout exposures (6 h/day x 3 days) to diesel exhaust (0.5 and 3.6 mg/m3) in whole-body inhalation chambers with or without particulates filtered. Significant bradycardia and T-wave depression were observed, regardless of the presence of particulates. Pulmonary inflammation was present only in the whole exhaust-exposed animals at the highest concentration. Fresh diesel exhaust or air was bubbled through the physiologic saline tissue bath prior to experiments to enable the isolated tissue exposure; exposed saline contained elevated levels of several volatile carbonyls and alkanes, but low to absent levels of polycyclic aromatic hydrocarbons. Vessels were then assayed for constrictive and dilatory function. Diesel components enhanced the vasoconstrictive effects of endothelin-1 and reduced the dilatory response to sodium nitroprusside. These data demonstrate that nonparticulate compounds in whole diesel exhaust elicit ECG changes consistent with myocardial ischemia. Furthermore, the volatile organic compounds in the vapor phase caused enhanced constriction and reduced dilatation in isolated coronary arteries caused by nonparticulate components of diesel exhaust.     

Stimulation of rat alveolar macrophage fibronectin release in a cadmium chloride model of lung injury and fibrosis.

Rats were exposed to saline or cadmium chloride (CdCl2) at 25, 100, or 400 micrograms/kg body weight by intratracheal instillation. At 3, 7, 14, and 28 days after exposure five animals/treatment were euthanized, the lungs were lavaged, and bronchoalveolar lavage fluid (BALF) was analyzed for lactate dehydrogenase (LDH), total protein, N-acetylglucosamindase (NAG), and cell number, type, and viability. Lung hydroxyproline concentration was characterized as a marker of lung collagen. Alveolar macrophages (AM) obtained in BALF were cultured and the release of fibronectin and TNF was determined. Lung tissue was examined microscopically at 28 and 90 days after exposure. Exposure to CdCl2 resulted in lung injury and inflammation demonstrated by increases in BALF LDH, total protein, NAG, and inflammatory cells. AM TNF release was not significantly changed by CdCl2 treatment. All doses of CdCl2 stimulated AM fibronectin secretion, a response which persisted throughout the 28-day postexposure period examined. Pulmonary fibrosis was demonstrated biochemically and/or histologically (trichrome staining tissue) at all CdCl2 dose levels. The association of CdCl2-induced AM fibronectin release with lung fibrosis confirms and extends previous observations relating AM-derived fibronectin to the development of interstitial lung disease and provides further evidence that the persistent increase in AM fibronectin release represents an early indicator of fibrosis.     

Labeling Rat Alveolar Macrophages with Fluorescent Microspheres

Abstract

Rats were exposed for various periods (5, 20, 60, and 120 min) to an aerosol of fluorescent polystyrene microspheres (FPM) with nominal diameter 1.07 μm. All the animals were killed 40 h later and the lungs were lavaged 10 times with physiological saline. Washings 1–2 and 3–10 were combined. The numbers of alveolar macrophages (AM) recovered by lavage were measured with a Coulter counter. Cytospin slides of cells in the lavage fluid were prepared and used to determine the fraction of recovered AM that contained FPM (labeling index, LI) and the FPM/AM profile. The same parameters were also measured by flow cytometry. There was good agreement between determinations of LI and FPM/AM by manual scoring methods and by flow cytometry. Samples of lavage fluid were digested with sodium hypochlorite (bleach), and aliquots of the resulting digests were filtered. Examination of the filters with epifluorescence microscopy enabled the total numbers of FPM present in the various lung washes to be determined. The lavaged lungs were also digested with bleach, and the number of FPM remaining was estimated by manual scoring only. Flow cytometry could not be used for this purpose, due to the presence of cellular debris. After exposure to airborne FPM at the concentration used for 2 h, less than half the recovered AM contained FPM. For studies of AM kinetics it will be necessary to achieve higher LI values and numbers of FPM per cell than were obtained in the present study.     

Decreased number of sperms and Sertoli cells in mature rats exposed to diesel exhaust as fetuses

This study was conducted to follow up the effects of fetal exposure to diesel exhaust on testicular cell numbers and daily sperm production in adulthood. Thirty-six pregnant rats were divided into five groups: groups exposed to total diesel-engine exhaust containing 1.71 mg/m3 particulate matter and 0.80 ppm nitrogen dioxide (high dose) or 0.17 mg/m3 particulate matter and 0.10 ppm nitrogen dioxide (low dose); groups exposed to filtered exhaust without particles containing 0.80 (high dose) or 0.10 (low dose) ppm nitrogen dioxide; and a group exposed to clean air. Exhaust exposure was performed from gestational day 7 to delivery. The numbers of daily produced sperm, spermatids and Sertoli cells in the diesel-exhaust-exposed groups were significantly lower than those in the control group on day 96 after birth. The ratio of spermatids/Sertoli cells and the follicle-stimulating hormone levels in the exposed groups were significantly higher. The present study provides evidence for the first time that mature rats exposed to diesel exhaust during fetus show a decrease in the daily production of sperm due to an insufficient number of Sertoli cells. As both the exhaust-exposed groups showed almost the same reactions toward the inhalation, the gaseous phase must have included the responsible toxicants.