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Pulmonary Effects of Ultrafine and Fine Ammonium Salts Aerosols in Healthy and Monocrotaline-Treated Rats Following Short-Term Exposure. F.R. Cassee, J. Arts, P. Fokkens, S. Spoor, A. Boere, L. van Bree, J. Dormans. Inhalation Toxicology, 14, 1215-1229, 2002. This is a study of a short term nose-only inhalation exposure of rats to model components of secondary PM (ambient fine particle air pollution) including ammonium bisulfite, ammonium ferrosulfate, and ammonium nitrate. Both normal rats and rats compromised with MCT (monocrotaline) were used. Secondary components of PM are inorganic and mainly nitrates and sulfates while primary components are elemental carbon and other organic compounds. Carbon black (CB) combined with ammonium nitrate is a model of primary PM and also studied for toxic effects on the lung as measured by analysis of BALF and histopathology. The concentrations of particles chosen for study were at least 10 times ambient levels. These levels were considered realistic for interpolation to human exposures by the investigators. The authors state two specific hypotheses:
Four specific questions were asked about PM:
Study Design: Six to 8 wk old SPF male Sprague Dawley rats were maintained under standard conditions. Twenty days before exposure to test atmospheres, 60 mg/kg MCT was administered. Exposure was for 4 hr/day and 3 consecutive days by nose-only inhalation. Twenty-four hr after the end of the last exposure day, animals were killed and prepared for determination of endpoints. The total number of animals used in the experiment is not specified. Generation and analysis of the 8 various test atmospheres of secondary PM were described. The atmospheres included ferrosulfate, nitrate, and 2 groups of bisulfate. Each of these 4 groups had a fine and ultrafine group within it with mass concentration (ug/m3), particle diameter (CMD and MMD), and number concentration (cm-3) presented as measured. Three carbon black/nitrate (0.6 um, 2 –9 mg/m3 carbon with ammonium nitrate of 0.4 – 0.18 mg3) atmospheres were generated and the same measurements made. Various measurement techniques are explained in the paper. SEM determined size and morphology of particles. A scanning mobility particle sizer (SMPS) was used to determine particle size distribution and numbers. The same rat was used for BALF studies and histopathology. The right lungs were lavaged for BALF while the left bronchus was tied off. LDH, N-acetylglucosaminidase (NAG) and albumin were measured in the BALF fluid. Total cell number, viability and differentials were also performed on BALF cells. Standard 5 um paraffin sections stained with both H and E and PAS-Alcian Blue were prepared for histopathology studies from the right lung. Results: The actual concentrations of UF and F secondary aerosols are presented in detailed tables in the manuscript as described in the study design section above. Mass concentration of UF particles ranged from as low as 70 ug/m3 in the bisulfate I group to 418 ug/m3 in nitrate group while the mass concentrations of fine particles ranged from 275 ug/m3 in the bisulfite I group to 407 ug/m3 in the bisulfite II group. Both CMD and MMD ranged from 41 to 107 nm in all 4 groups of ultrafine secondary particles while these parameters ranged from 299 to 643 nm in all 4 groups of fine particles. Finally the number concentration ranged from 1.7 x 103 to 4 x 103 cm–3 in the UF groups and 1.8 x 103 to 9.2 x 103 in the F groups. The geometric diameter of UF was 0.04 – 0.12 um and of F was 0.20 – 0.60 um as determined by SEM of ammonium particles. CB particles were observed by SEM to be about 0.03 to 0.07 um and spherical. The 3 groups of CB/nitrate aerosol had much larger mass concentrations than did the F or UF secondary aerosol groups. The CMDs and MMDs of the CB/nitrate groups were similar to those of the fine secondary aerosols. Observation of the experimental animals showed no abnormalities in either the MCT-treated rats, control rats or those exposed to test atmospheres. MCT treated rats had increased lung weights and decreased liver and heart weights. The authors state that there no test aerosol related changes were observed in body or organ weights although these data are not shown. BALF results were presented in 5 to 6 rats in each of the 4 secondary particle groups. Protein, LDH and NAG were measured in sham-exposed animals, UF exposed and F exposed animals within each of the 4 types of atmospheres. PM exposure had minimal effect on these endpoints in healthy animals. All indicators of toxicity were higher in MCT treated animals. High protein and albumin levels were seen in MCT treated rats. These levels were slightly higher in rats exposed to fine ammonium nitrate. MCT treated rats all had increased numbers of white cells including foamy macrophages and monocytes, neutrophils, and lymphocytes in the BALF. MCT also caused medial hypertrophy of the pulmonary arteries and neomuscularization of the small blood vessels. None of the increases were related to exposures. Similarly pathological changes in the lungs were related only to MCT. Changes were seen in the pulmonary arteries, small blood vessels, alveoli which containing foamy macrophages, and liver with periportal glycogen deposits. The authors state that there was no change in histology related to aerosol exposure in any animals. At the end of the results section, the authors note that there was a background infection of Hemophilus sp in the experimental rats. Exposure to most of the aerosol groups did not cause any morphological changes in the lungs of control (not treated with MCT) rats. However, according to the authors, histopathological lesions in both F and UF ammonium nitrate aerosol due to the infection were more frequent and stronger compared to their controls. Light microscopic exam of the CB/nitrate group showed increased black material in alveolar macrophages. Pathological changes found in all animals are described at the end of the results section. These include perivascular inflammation, alveolar edema, and hypertrophy of the bronchiolar epithelium. The authors speculate that these changes may be due to the influenza infection. Discussion: The authors address their initial goals in the discussion. They also give a thorough discussion of their results as related to the literature on F and UF particles and combinations of such. They conclude that exposure of up to 400 ug/m3 ammonium salts does not cause adverse effects in healthy or MCT treated rats. Similarly exposure to ammonium nitrate and CB does not cause adverse pulmonary effects. Different particle sizes do not cause different effects in normal or MCT treated rats. The investigators do note that the Hemophilus infection could have overshadowed an adverse effect. Finally they speculate that the nitrate stimulated growth of bacterial infection because the pathological lesions were more frequent in the nitrate group. It was concluded that the MCT model was not useful at low levels of particulates because it caused large variations in the indicators of lung injury. Hence any small but statistically real variations could be missed in the data. The lack of adverse pulmonary effects as measured by the parameters in this study does substantiate studies by other laboratories with the same compounds at high levels. The authors speculate that the sulfates and nitrates may be neutralized by ammonia and thus harmless at ambient levels. In regard to the second goal of this study, neither particle size nor number concentration could be determined to have more importance in effecting measured parameters. Thus it was not possible to rank F and UF ammonium salts by toxicity. The investigators conclude that no adverse pulmonary effects were caused by the exposure regimens used in this study.
Editorial opinion: This was a very ambitious experiment involving large numbers of rats and multiple treatment groups. The particles and levels used were environmentally relevant. The study also incorporated a population of rats representing healthy individuals and a population representing individuals with comprised heart and lung function (MCT treated). In an attempt to gain data relevant to human exposures, the concentrations of F and UF particles used were relatively close to ambient levels. For readers interested in the details, reading the paper and examining the tables will be very helpful. There are two points in the study which could be emphasized. First, the SPF rats used developed a respiratory tract infection (Hemophilus or influenza). This was not reported until the end of the results. Since all rats appeared to be affected, the infection was probably incurred between the time the animals were received and treated with MCT (20 d) and when they were exposed to the particle regimens. However, this study does contain useful data because none of the exposure regimens appeared to cause adverse affects 24 hr after the last exposure. One might have expected infected animals to show more toxicity from particles than healthy animals. This did not occur and the authors were able to conclude that no adverse pulmonary effects were caused by the experimental protocol. They also fairly discuss the implications of having sick animals in the study. In fact this study could be considered a model for a worst case scenario of exposure to F and UF particles. The second point that that is not obvious in reading is that these animals were exposed by nose-only exposure. This technique is becoming more common in experimental use but is not equivalent to a whole body exposure. Thus comparison of results from nose-only exposure to those from whole body exposure may not be completely justified. One of the first reviews in this newsletter concerns advantages and disadvantages of nose-only exposure and can be found at: Nose Only Directed Flow Systems, Principles and Advantages.
By: Susan G. Shami, ScD, Editor
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