Irreversible pulmonary changes induced in rat lung by dust overload.

The objective of this study was to investigate whether the effects of dust overload are reversible upon cessation of subchronic exposure to test toner. Female rats were exposed 6 hr/day, 5 days/week for 3 months to a test toner at 0, 10, and 40 mg/m3. The retained quantity of test toner in the lungs at the end of exposure was 0.4 and 3.0 mg for the low and high exposure groups, respectively. Fifteen months later, the corresponding values were 0.12 and 2.65 mg in the lungs. Alveolar clearance of tracer aerosols as well as cytologic and enzymatic parameters in the bronchoalveolar fluid was investigated at the end of exposure and subsequently up to 15 months later. The alveolar clearance of 59Fe2O3, 51Cr-polystyrene, and 85Sr-polystyrene tracer aerosols was slightly retarded at the low and substantially impaired at the high exposure level. At the low exposure level, there was some recovery in the clearance behavior up to 6 months after exposure. In contrast, at the high exposure level there was no indication of a reversal of the impaired clearance. For the beta-glucuronidase activity and the number of polymorphonuclear cells, the pattern of the effects was similar to the effects on the half-time tracer particle clearance. In conclusion, the dust overload at a lung burden of 3 mg test toner in rats was persistent for at least 15 months after termination of exposure.


Introduction
An impairment of alveolar clearance was observed after exposure to a high concentration of insoluble particles in previous subchronic and chronic inhalation studies in rats (1,2). In the same chronic study, an increase ofpolymorphonuclear neutrophils (PMN) in the bronchoalveolar lavage fluid (BALF) indicated inflanmmatory responses in the lung (3). These effects are some characteristic signs oflung overloading, a phenomenon that has been discussed by Morrow (4). The objective ofthe present study was to investigate whether these effects are reversible upon cessation of exposure. After a subchronic inhalation of a pigmented polymer (test toner) during a 15-month post-treatment observation period, the clearance oftracer particles, the number ofcells in the BALF, and the level ofsome characteristic enzymes in the BALF were measured.

Materials and Methods
A 9000-type xerographic toner material composed of about 90% styrenell-butylmethacrylate random copolymer (CAS no. 25213-39-2) and 10% high-purity furnace-type carbon black (CAS no. 7440-44-0) was specially prepared for animal studies. Relative to commercial toner, the respirable fraction of particles was enriched about 10-fold according to the American Conference ofGovernmental and Industrial Hygienists criteria. The mass median aerodynamic diameter (MMADD) was 4.0 jAm with a geometric standard deviation of 1.5. Female SPF F-344 rats were exposed 6 hr/day, 5 days/week for 3 months to this test toner at aerosol concentrations of0, 10, and 40 mg/mi3. The retention of test toner in the lungs and in the lung-associated lymph nodes (LALN) was analyzed by a photometric determination after dissolving the lung tissue (1).
To allow a better insight into exposure effects on pulmonary clearance kinetics, y-labeled particles were also administered which permit noninvasive measurements. The tracer aerosols of Pe2O3, 5tCr-polystyrene, and tm5Sr-polystyrene with MMADDs ofabout 0.4, 0.76, and 3.8 ism and geometric standard deviations of 1.9, 1.5, and 1.26, respectively, were inhaled (nose only) for 0.5-1.0 hr by eight animals per group (2). The retained lung burden oftracer particles was below 1 pg. The thoracic Zy activity was measured twice weekly. The rate coefficients of alveolar clearance were calculated from data for days 15-90 for each animal. After the 90-day period, animals were sacrificed and the 'y activity ofthe lungs, the LALN, and the BALF was measured.
The BALF was obtained by a first set of lavages with 2 x 4 mL saline and a second set of lavages with 4 x 5 mL saline and mild massage of the lung. A differential cell count of the BALF enabled the number of macrophages, polymorphonuclear cells (PMN), and lymphocytes to be determined. The distribution of test toner particles in macrophages was analyzed by light microscopy. After centrifugation of cells, lactate dehydrogenase (LDH), ,3-glucuronidase, and total protein were measured in the supernatant.
At 3 months and 18 months of the study, five rats from each absolute values are presented only forthe control group, wheregroup were sacrificed for histopathology. Methods of animal as for all exposure groups values are shown as percentages ofthe sacrifice, tissue processing, and histopathology are described by control values. In the high exposure group, LDH, ,8-glucuroni-Muhle et al. (3).
dase, and total protein were significantly elevated, and only a minor recovery was observed 15 months after termination ofex-Results and Discussion posure. For LDH and ,l-glucuronidase, the highest increase compared to controls was detected after 3 months. The differen-The quanitity of test toner retained in the lungs at the end of tial cell count indicated only a slight increase in the number of exposure was 0.4 and 3.0 mg for the low and high exposure PMN in the low exposure group but a substantial increase after groups, respectively. After 15 months, the corresponding values high exposure. The number of lavagable macrophages were were 0. 12 and 2.65 mg in the lungs and about 5 and 190 ,g in the only slightly affected in both exposure groups. LALN. From the retention data, overall half-times of toner The alveolar clearance of all three 'y-labeled particles was clearance were calculated as 277 and 2845 days for the low and slightly retarded at the low exposure level, but almost completehigh exposure groups, respectively.
ly impaired at the high exposure level. The most pronounced im-The results ofBALF enzyme analysis, differential cell count, pairment of the alveolar clearance of the polystyrene tracer and tracer clearance are summarized in Tables 1 and 2. The particles occurred 3 months after termination of exposure to the test toner. Thereafter the clearance impairment was reduced from a factor of about 10 to a factor of4 at 12 months compared to control values. In the high exposure group, the pattern ofthe effects during the 15-month post-treatment observation period was similar for (glucuronidase activity, half-time of tracer polystyrene particles, and number ofPMN in BALF. The observation ofthe persistent retardation of the alveolar clearance, even after a 12-month recovery period, was unexpected. During this recovery period, the particle distribution in the lung is changed. Histopathologically, aggregations ofparticle-laden macrophages were detected in focal areas, whereas in most areas ofthe lung, particle-laden macrophages were found only sporadically. The half-time for particle clearance ofa few hundred days is much longer than the turnover time of an alveolar macrophage, which was estimated to be about 4 days in mice under the condition ofno particle load (5). This means that there is a release of particles by dying macrophages and an immediate rephagocytosis by intact or newly arrived macrophages. At the high exposure level the percentage of macrophages without toner particles in the BALF increased from 25% at the termination ofexposure to 85% after the 15-month observation period. Nevertheless, these macrophages were not able to remove recently inhaled tracer particles. A possible explanation is a release of chemotactic factors from particle-laden macrophages in areas of sequestration, which induce other macrophages containing labeled particles to remain in the vicinity of this area instead of being cleared to the upper respiratory tract.