Effect of concentration and cumulative exposure of inhaled sulfuric acid on tracheobronchial particle clearance in healthy humans.

We have previously shown that 1-hr exposures to 0.5 microns sulfuric acid (H2SO4) mist at 100 and 1000 micrograms/m3 produced transient alterations of bronchial mucociliary clearance of monodispersed 7.6 and 4.2 microns mass median aerodynamic diameter gamma-tagged ferric oxide (Fe2O3) in healthy nonsmoking humans in a dose-dependent manner. To determine the role, if any, of the length of exposure, 10 healthy volunteers were exposed to 100 micrograms/m3 H2SO4 for 1 hr and 2 hr on separate occasions, 1 week apart, with measurements of their mucociliary clearance of 5.2 microns Fe2O3 particles inhaled both before and after the inhalation of the H2SO4. Their rate of bronchial mucociliary clearance was markedly reduced for both Fe2O3 aerosols, with slower clearance of the aerosol inhaled after the H2SO4 exposure. For the tagged Fe2O3 aerosol inhaled after exposure for 2 hr at 100 micrograms/m3 H2SO4, the tracheobronchial clearance halftime, (T50), tripled from control, and the reduced rate of clearance was still evident 3 hr after the end of exposure. The 1-hr 100 micrograms/m3 H2SO4 exposure doubled T50 from control, and the reduced rate of clearance lasted for about 2 hr after the end of exposure. These results indicate that the effect of doubling the length of exposure was as great or greater than an order of magnitude increase in the concentration of H2SO4.


Introduction
Mucociliary clearance is a lung defense mechanism by which inhaled particles that deposit on the conducting airways are removed from the lung. This removal is by a coordinated mucociliary transport system that relies on the beating of the cilia to move the overlying mucus layer carrying the deposited debris.
While the mechanisms by which acid aerosols affect mucociliary clearance are not well understood, it is still important to determine the response of the mucociliary system to these challenges, in particular at concentrations and time periods relevant to environmental and occupational exposures. This paper describes studies on the effect of concentration and length of exposure to inhaled sulfuric acid (H2SO4) aerosols on tracheobronchial particle clearance in humans. Studies on the response to acid aerosols on lung clearance in animals has already been discussed at this symposium by R. Schlesinger (1).
Our previous results on the effect of H2SO4 concen-*New York University Medical Center, Institute of Environmental Medicine, 550 First Avenue, New York, NY 10016. tration on mucociliary clearance in humans are shown on Figure 1. Ten healthy volunteers inhaled 0.5 ,um H2SO4 for 1 hr at concentrations of 100 and 1000 ,ug/rm3. When the tracer aerosol was 7.6 gm in aerodynamic diameter, the response shown in Figure  lA indicates faster clearance at the lower exposure concentration and slower clearance at the higher concentration. In Figure 1B, eight healthy volunteers had the same exposures (100 and 1000 gg/m3 of 0.5 gm H2SO4 for 1 hr), but here the tracer aerosol was only 4.2 gm in aerodynamic diameter; the response at the higher concentration is to slow clearance as before, but for 100 gg/m3, clearance is slower than in the control test.
This apparent contradiction has to do with the fact that the mucociliary clearance technique depends on the deposition pattern of the inhaled, tagged aerosols. The larger 7.6 ,um particles deposit much more in the large central airways than the 4.2 gm particles, and therefore they will indicate clearance preferentially from a lung region where the effective H2SO4 deposited dose is quite small. Thus, it seems that low H2SO4 exposure concentrations stimulate mucociliary transport. On the other hand, at high concentrations, the delivered dose is increased, slowing down the mucociliary clearance in TC A. 1  inhaled the aerosol through a nasal mask while sitting quietly in order to simulate normal human exposure conditions. The H2SO4 droplets had a mass median aerody-.~~~^_ namic diameter (MMAD) of 0.5 gm and a geometric standard deviation (6g) of 1.9. The electrostatic charge I I I of the aerosol was less than 20 charges per droplet. The 4 5 6 air temperature and relative humidity during the 1-hr exposures were 27.1 ± 0.60C and 47 ± 2% (mean ± SD), respectively. The H2SO4 concentrations deterto 0 (control), 100, and mined from the filter samples collected during expo-Dnchial clearance. (A) sure were < 5; 104 ± 21 and 111 ± 22 ,ug/m3 for the LM tracer aerosols. control, 1-hr (short) and 2-hr (long) exposures, respec-.2 gim MMAD tracer tively, and there were no significant differences in particle size, temperature, or humidity between different exposures. a dose-dependent manner. Similar responses were obtained from a group of six asymptomatic asthmatics that were exposed for 1 hr to 100, 300, and 1000 ,ug/m3 H2SO4 (2). This paper reports the effects of exposure duration of 100 tg/m3 on mucociliary clearance in healthy, nonsmoking adult volunteers.

Subjects
Ten adult male volunteers participated in the study. Each subject gave informed consent; completed a questionnaire on pulmonary disease, smoking, and work history; and underwent a clinical examination which included chest roentgenogram, electrocardiogram, and blood and urine analyses. All protocols were

Respiratory Mechanics
The possible effects of H2S04 aerosol upon respiratory mechanics were investigated by having each subject perform a series of respiratory function tests prior to exposure, within 15 min after the cessation of exposure, and again 3 hr later. Subjects performed the tests while wearing a nose clip in a sitting position, and all parameters were standardized to body temperature, pressure, and water saturation (BTPS). A series of maximum expiratory flow-volume maneuvers yielded forced expiratory volume at 1 sec (FEV1), forced vital capacity (FVC), mid-maximal expiratory flow rate (MMEF), and peak expiratory flow rate (PEFR). These measurements were performed with a low-resistance (< 0.2 cm H20/L/sec) wedge spirometer (Model 170, Med-Sciences Electronics, Inc., St. Louis, MO), a waveform recorder (Model 1015, Biomation, Inc., Cupertino, CA), and plotted with an X-Y-T chart recorder (Model 2000, Houston Instruments, Austin, TX). Values were expressed as a mean of three tracings in which the FVC was within 95% of the best effort. Thoracic gas volume (Vtg), airway resistance (Raw), and airway conductance Gaw (the inverse of Raw) were determined by body plethysmography. Tests were conducted with a 600-L stainless-steel body plethysmograph and associated electronics (Model 2000, Cardio-Pulmonary Instruments Corp., Houston, TX). Measurements were made approximately 3 min after each subject entered the plethysmograph to allow for thermal equilibrium. Values were expressed as the means of five panting maneuvers after subtracting either the measurements shutter assembly dead space (35 mL) or the equipment resistance (0.28 cm H20/L/sec).
Single-breath nitrogen washout tests were performed with a nitrogen analyzer (Model 1410, W. Collins, Inc., Braintree, MA). Volumes were measured by the integration of the expiratory airflow as measured by a heated pneumotachograph (No. 3, A. Fleisch, Switzerland). The slope of the alveolar plateau of phase III (A%N2/L), closing volume (CV), and the ratio of closing volume to vital capacity (CV/VC) were determined.

y-Tagged Ferric Acid Aerosols
The possible effect of H2SO4 dose on mucociliary clearance was studied by monitoring two inhaled monodispersed y-tagged aerosols. Immediately before the acid exposure, the subjects inhaled, via a mouthpiece, a monodispersed Fe2O3 aerosol for about 2 min. This aerosol, tagged with the y-emitter gold-198 (198Au, EY= 410 keV, t1/2 = 2.7 days) was generated with a spinning disc generator. Immediately after the acid exposure, the second aerosol, tagged with technetium-99m (99mTc, EY = 144 keV, t1/2 = 6 hr) and generated as the first one, was inhaled. All inhalations were at respiratory rate, 14.1 ± 0.2 bpm; tidal volume, 1.09 ± 0.31 L; peak inspiratory flow, 69.4 ± 11.2 Lpm: average inspiratory flow, 40.8 ± 7.9 Lpm: inspiratory time, 1.70 ± 0.2 sec. The diameter of the tagged aerosols were chosen to produce a ratio of about 70% tracheobronchial, 30% alveolar initial deposition. This was determined, for each subject, on a test run in which their baseline mucociliary clearance was measured using simultaneously two aerosols of about 3.5 ,um and 6.0 gm aerodynamic diameter tagged with 198Au and 99mTc, respectively. From this run, the subject's optimum particle aerodynamic diameter was determined; the group mean was dA = 5.2 ,um, with a range of 4.0 to 5.8 ,um.

Measurement of Mucociliary Clearance
Lung clearance was determined from serial measurements of right lung retention, using two large Nal (T1) collimated scintillation detectors aligned over the upper right lung field and connected to a preamplifier-amplifier-single channel analyzer-interface-Apple II microcomputer. The single channel analyzers energy discrimination windows were set for the 99mTc and the 198Au peaks. The Apple II was programmed to accept the counts, correct them for background and decay, and to display the corrected retention curves in real-time on the screen. All y-retention measurements were taken inside a low-background chamber.
Depending upon the position of the subject relative to the detectors, the viewed field included either the whole head or approximately 90% of the right lung. The detectors were first aligned over the subject's head for a measurement of the initial amount of aerosol deposited in the upper respiratory tract (URT). The subject was then repositioned so that the detectors viewed the right lung. After the first measurement, used to determine the initial amount of tagged aerosol deposited in the lung, the subject breathed from an air stream containing either the H2SO4 aerosol at the preset concentration or the distilled water control aerosol. Serial measurements of lung retention were made during the next 5 hr. The same procedure to determine URT deposition and initial lung deposition was done after the H2SO4 exposure for the second tagged aerosol. All subjects returned for an additional retention measurement the following morning.
Tracheobronchial retention curves were constructed in which the initial tracheobronchial retention value was 100% and the 24-hr thoracic retention value was 0%. As previously, the 24-hr thoracic retention was considered an indicator of the deposition in the nonciliated pulmonary or alveolar region (ALV). For each run, we obtained two tracheobronchial clearance curves, one starting immediately before the H2SO4 inhalation and continuing for about 5 hr, and another with initial time at the end of the acid exposure. From each clearance curve we obtained the following characteristic tracheobronchial clearance times: T50, the time to complete 50% of tracheobronchial clearance; and MRT, the mean residence time for particles on the tracheobronchial airways.

Protocol
The experimental protocol followed a similar sequence to our previous studies on the effect of H2SO4 on healthy and asymptomatic asthmatics subjects (3,4), with the addition of a second tagged aerosol inhalation following the end of the period of inhalation of the H2SO4. A baseline respiratory function test preceded a 2-min 198Au-Fe203 test aerosol inhalation during each run day, followed by 3-min head and initial lung retention measurement. This was immediately followed by three 20-min inhalations for the short (1-hr) exposure, or six 20-min inhalations for the long (2-hr) exposure, either a distilled water sham aerosol, for the control runs, or 100 jg/m3 of 0.5 jim diameter H2SO4. Between the three or six inhalation periods, 3-min lung retention values for the 198Au-aerosol were recorded and, at the completion of the H2SO4 exposure, the second tagged aerosol, 99mTc-Fe2O3, was inhaled for 2 min and head and initial 99mTc chest counts were recorded. Thereafter, simultaneous measurements of retention were performed for both 198Au and 99mTcaerosols. After accruing 1 hr of retention data, the second set of respiratory function tests was obtained. Another 30 min of retention measurements preceded a 30-min lunch period, followed by more retention measurements to complete the 5-hr period from the initial 198Au-aerosol inhalation. Before the end of the day, the last set of pulmonary function tests were performed.

Respiratory Functions
The respiratory mechanics of the 10 subjects were not significantly affected by the exposure to H2S 04 at a concentration of 100 jig/m3 for neither the short 1-hr exposure nor for the long 2-hr exposure.
Regional Deposition of Tagged Aerosols Regional shifts in the deposition of the tagged aerosol can create artifacts in the thoracic retention curves, and parameters such as size distribution of the aerosol and breathing pattern during inhalation must be controlled within runs. The measurements of regional deposition for the group (i.e., initial upper respiratory tract, URT; tracheobronchial retention, TB; 24-hr thoracic retention as an indicator of the deposition in the nonciliated pulmonary or alveolar region (ALV), are presented in Table 2. There were no significant differences between the values for the control and for the H2SO4 exposure runs neither when the tagged aerosol was inhaled before the H2SO4 nor when it was inhaled after acid exposure. If there was any variation in regional deposition, it was not large enough to alter the apparent rate of clearance of the Fe203 aerosol.
Bronchial Mucociliary Clearance Table 3 shows that the alterations in bronchial clearance after inhalation of H2S 04 were statistically significant. For 1-hr and 2-hr exposures at 100 jig/m3 H2SO4, the average clearance halftimes, T50, were significantly greater than those following the sham exposure, indicating a delay in mucociliary clearance. The effect of H2SO4 on clearance was greater when the tagged aerosol was inhaled after the acid dose exposure. A similar result is indicated by the effects on mean residence time, MRT. The group mean MRT values increased after 1 hr of H2SO4 exposure, and increased even more after 2 hr of exposure.

Discussion
Bronchial mucociliary clearance was altered by 100 jg/M3 H2SO4 exposure in a group of 10 healthy subjects in a dose-dependent manner, similar to that established in this laboratory for H2SO4 concentrations of 100, 300, and 1000 jig/m3 for 1-hr, (3,4). There was a transient delay in mucociliary clearance as defined by higher values of T50, increasing with longer exposure times. The differences were statistically different from control (p < 0.05) for both the 1-hr and 2-hr exposures. Figure 2 shows the mean clearance for the group, for each exposure, and for the control run.
These results are analogous to our previous results ( Fig. 1), where the subjects inhaled H2SO4 for 1-hr at 100 and 1000 jig/m3. The magnitude of the effects on T50 and MRT are summarized in Table 4. For the 1-hr 100 jig/m3 H2SO4 exposure, we obtained a similar increase in T50 for the eight healthy subjects that inhaled a 4.2 jim tagged aerosol before the H2SO4. The increase in T50 after 2 hr of 100 jig/m3 H2SO4 exposure is quite close to our previous 1 hr 1000 jig/m3 H2SO4. These results show that increasing the time of exposure  of time for which we have actual data. By inspecting the clearance curves for control and exposure runs, we can see that increasing the integration period will only increase the AMRT values. We have previously shown in this laboratory in donkeys that there is a time period, of the order of 30 min, for the effect of an acute H2SO4 exposure to appear as an alteration of mucociliary retention curves (5). When comparing the retention curves of inhaled tagged aerosols before and after the same H2SO4 exposure (Fig. 3), it becomes noticeable that both curves become parallel to each other after about 40 min, indicating the same rate of clearance of particles from the lung. 100 Control L Short Acid  The magnitude of an effect can be characterized by the amount of change produced and the duration of this a eration for a given end point. In the present study, changes are defined by the variations in the rate of clearance of tagged particles from the lung. Figure 4 shows the percentage change (from control) of clearance halftime of aerosols inhaled after 1-hr and 2-hr exposures to 100 ,ug/m3 H2SO4, as a function of time after the end of the H2SO4 exposure. The two exposures yield very different rates of clearance on the same time scale. After the 1-hr H2SO4 exposure, tracheobronchial clearance slows down for about 2.5 hr and, even though the amount cleared is only at this time about 50% of that in control runs, the rate of clearance has reached the level of that in control runs. For the 3 hr following the 2-hr H2SO4 exposure mucociliary clearance is slowed to -50%/hr of that in the control test, and there is no evidence of it leveling off.
In conclusion, the duration of an acute H2S 04 exposure to 100 ,ug/m3, affects mucociliary clearance not only in magnitude but also in the persistence of the changes.