ToxSci Advance Access originally published online on July 13, 2006
Toxicological Sciences 2006 93(2):400-410; doi:10.1093/toxsci/kfl059
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Published by Oxford University Press 2006.
Particle Deposition in Spontaneously Hypertensive Rats Exposed via Whole-Body Inhalation: Measured and Estimated Dose
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* Department of Environmental Sciences and Engineering, School of Public Health, University of North Carolina, Chapel Hill, North Carolina 27599; Environmental Media Assessment Group, National Center for Environmental Assessment, MD B243-01, and Pulmonary Toxicology Branch, Experimental Toxicology Division, National Health and Environmental Effects Research Laboratory, Office of Research and Development, U.S. Environmental Protection Agency, Research Triangle Park, North Carolina 27711; and Office of Research and Development, U.S. Environmental Protection Agency, Research Triangle Park, North Carolina 27711
1 To whom correspondence should be addressed. Fax: (919) 541-1818. E-mail: wichers.lindsay{at}epa.gov.
Received April 20, 2006; accepted July 9, 2006
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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A plethora of epidemiological studies have shown that exposure to elevated levels of ambient particulate matter (PM) can lead to adverse health outcomes, including cardiopulmonary-related mortality. Subsequent animal toxicological studies have attempted to mimic these cardiovascular and respiratory responses, in order to better understand underlying mechanisms. However, it is difficult to quantitate the amount of PM deposited in rodent lungs following inhalation exposure, thus making fundamental dose-to-effect assessment and linkages to human responses problematic. To address this need, spontaneously hypertensive rats were exposed to an oil combustion–derived PM (HP12) via inhalation while being maintained in whole-body plethysmograph chambers. Rats were exposed 6 h/day to 13 mg/m3 of HP12 for 1 or 4 days. Immediately following the last exposure, rats were sacrificed and their tracheas and lung lobes harvested and separated for neutron activation analysis. Total lower respiratory tract deposition ranged from 20–60 µg to 89–139 µg for 1- and 4-day exposures, respectively. Deposition data were compared to default and rat-specific estimates provided by the Multiple Path Particle Deposition (MPPD) model, yielding model predictions that were < 33% of the measured dose. This study suggests that HP12 exposure decreased particle clearance, as the mass of HP12 in the lungs following a 4-day protocol was nearly four times that observed after a 1-day exposure. This work should improve the ability of risk assessors to extrapolate rat-to-human exposure concentrations on the basis of lung burdens and, thus, better relate inhaled doses and resultant toxicological effects.
Key Words: rat; particle; deposition.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Human epidemiological studies have established that exposure to particulate matter (PM) can lead to adverse health effects involving the respiratory and cardiovascular systems (as reviewed in Brook et al., 2003
; Brunekreef and Holgate, 2002; U.S. EPA, 2004); these findings have largely been corroborated by animal toxicological research (Schlesinger, 1995; U.S. EPA, 2004; Watkinson et al., 2001). However, in spite of extensive efforts to determine the modes of action for PM, a number of uncertainties remain regarding the mechanisms by which PM exerts its effects.
Investigators conducting PM research commonly employ exposure routes of intratracheal instillation, nose-only inhalation, and whole-body inhalation, with each technique having its distinct advantages and disadvantages (Costa et al., 2006; Dorato and Wolff, 1991; Driscoll et al., 2000). While it is clear that inhalation represents the most relevant physiological means of exposure to atmospheric PM, this exposure route does not readily lend itself to precisely relating target dose to toxic effect. Despite this limitation, a number of studies have been undertaken to determine aerosol deposition in unanesthetized rodents, primarily using nose-only exposure systems (Asgharian et al., 2003; Cassee et al., 2002; Chen et al., 1989; Newton and Pfledderer, 1986; Snipes et al., 1988). To date, there have been few laboratory animal studies involving inhalation exposure to PM that have collected respiration data in unrestrained, unanesthetized rats during the exposure period (Asgharian et al., 2003; Cassee et al., 2002), and these studies have generally employed monodisperse and/or ultrafine particles.
To meet the need for toxicological studies that better characterize PM dosimetry, an exposure system was developed which combined a dry particle string generator (Ledbetter et al., 1998) with whole-body plethysmograph (WBP) chambers (Wichers et al., in press). This novel setup permitted inhalation exposures within rat WBP chambers, such that breathing parameters could be collected concurrently with PM exposure. Thus, with this system, the requisite exposure and ventilation data, including chamber concentration, breathing frequency (f), and tidal volume (VT), were obtained for empirical and theoretical estimations of PM dose.
There have been a handful of publications that have employed the Multiple Path Particle Deposition (MPPD) model (Anjilvel and Asgharian, 1995) to compare estimates of deposition in humans and rats based on published data (Anjilvel and Asgharian, 1995; Brown et al., 2005; Cassee et al., 2002; Jarabek et al., 2005). In two rodent studies comparing experimentally obtained deposition values to MPPD model estimates, one found good accuracy (Anjilvel and Asgharian, 1995) and the other reported incorrect (Cassee et al., 2002) predictions of dose. Although the model outcomes for these studies incorporated rat-specific breathing parameters, the rats had been exposed to PM via nose-only inhalation. To our knowledge, there has been no evaluation of the model under whole-body inhalation conditions.
The main objective of this research was to determine the deposition of well-characterized, polydisperse particles delivered via whole-body inhalation in Spontaneously Hypertensive (SH) rats, in order to permit comparisons between previous rodent exposures and toxicological outcomes. A second objective of this study was to compare particle deposition between a single 6-h exposure and four consecutive daily 6-h exposures, the latter allowing 18 h for clearance between each exposure. Such comparisons would provide better characterization of the kinetics of particle deposition and retention. The final aim of this study was to compare measured deposition to that predicted by the MPPD model using both rat-specific and default inputs. It is expensive, in terms of time and resources, to measure deposition; therefore, it is impractical to assess dose in every PM inhalation study. However, once validated using rat-specific data, it should be possible to use the MPPD model to estimate inhaled dose in a variety of studies, thus providing important insights into dose-response relationships.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Animals.
Male SH rats were obtained from Charles River Laboratories (Raleigh, NC) and double housed in plastic cages with beta-chip bedding in an isolated room within an American Association for Accreditation of Laboratory Animal Care–accredited animal care facility. A 12-h light:12-h dark cycle was imposed from 0600 h–1800 h to 1800 h–0600 h daily. Rats were randomized and assigned to exposure groups based on initial body weight; at the start of each study, rats were between 71 and 73 days of age and weighed 255–278 g. Rats received laboratory feed (Purina rat chow; Brentwood, MO) and water ad libitum from time of receipt to time of sacrifice, except for the 6.5-h periods when they were maintained in the WBP chambers. Each morning and afternoon (pre- and postexposure, respectively), rats were reweighed to assess overall health.
Particles.
An oil combustion–derived PM (HP12) was employed in this study, and particle size was determined prior to exposure via a seven-stage cascade impactor (Intox Products, Albuquerque, NM). HP12 had a mass median aerodynamic diameter (MMAD) of 1.95 µm and a geometric standard deviation (GSD, 
g) of 3.49. The results from an initial chemical analysis of bulk HP12, assessing metal, sulfate, and carbon levels, are reported elsewhere (Wichers et al., 2004a). A more thorough analysis of HP12 was conducted at the conclusion of this study to identify whether resuspended HP12 was of the same chemical composition as the bulk sample (Wichers et al., in press). The composition of the resuspended HP12 was found to be in good agreement with the bulk sample, indicating that there was little change in particle chemistry attributable to grinding and/or aerosolization. The metals with the highest content in HP12 were V, Al, Fe, Ni, and Zn. Of these five elements, the latter four were partially soluble when extracted in deionized water and 1M HCl (Table 1); V, for the most part, was poorly soluble based on these same extractions.
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Exposure system.
Briefly, a string generator system (Ledbetter et al., 1998
) was used to resuspend dry HP12 for distribution to six WBP chambers. The system operated at a flow rate of 13 l/min and, with each exposure, the WBP chamber received approximately 2 l/min of filtered room air or PM aerosol. The remaining 1 l/min was routed to a sample port which was used to determine HP12 concentration once every hour during exposure using a 47-mm Teflon filter attached a flow meter. When the aerosol concentration was not being assessed gravimetrically (
55 min/h), a real-time aerosol monitor (Dust Trak; TSI, Inc., St Paul, MN) was used to roughly approximate concentration and make adjustments to the string generator when the particle concentration deviated from the target exposure concentration. Control WBP chambers were connected to an independent air system and supplied with 2 l/min of filtered room air. The resistance to the exhaust flow for each WBP chamber was adjusted daily such that the internal pressures within all chambers were equivalent but slightly negative with respect to ambient air. Daily individual WBP chamber PM masses were evaluated via a 47-mm Teflon filter attached in line to the exhaust of each WBP chamber. Complete details of the exposure system have been submitted for publication (Wichers et al., in press).
Experimental design and procedure.
This study was conducted in two phases of differing durations to assess particle retention and clearance. Phase I lasted 2 days, with all rats receiving filtered room air on the first day (day–1); rats in the control group were also given filtered air on the second day (day 0) while the exposure group received HP12 aerosol. Rats in phase II were similarly exposed to filtered room air on the control day (day–1) but then underwent 4 consecutive days of HP12 aerosol exposure (days 0–3); control rats received filtered air throughout the study. Immediately following the last exposure day (day 0 for phase I and day 3 for phase II), rats were sacrificed, and the tracheas and lung lobes were collected and separated for neutron activation analysis (NAA). The exposure regimens of 1 and 4 days were chosen such that the 1-day exposure minimized the time available for particle clearance and the 4-day exposure matched prior studies conducted in our laboratory (Campen et al., 2001). For the current work, the exposure duration was 6 h (0800 h–1400 h), and the target HP12 concentration was 13 mg/m3. The target concentration was selected based upon the exposure system capabilities, as well as the ability to foster comparisons with previous instillation studies (Wichers et al., 2004a,b).
Unanesthetized WBP, and analyses.
Whole-body plethysmography (WBP) was employed to obtain rat-specific data for use in the MPPD model; these data included f and VT, as well as time of inspiration (TI) and time of expiration (TE) used for the calculation of inspiration fraction (IF, where IF = TI/(TI + TE)).
Eight WBP chambers (Model PLY3213; Buxco Electronics, Inc., Sharon, CT) were used for both phases (six aerosol and two air). All chambers had been previously sprayed with a nonstatic solution (Staticide; ACL, Elk Grove Village, IL), which was used to minimize particle charge (and therefore loss) during exposures, and each chamber was calibrated daily just prior to animal loading. Rats were maintained in the WBP chambers for 6.5 h daily (0730 h–1400 h), with the first 30 min serving as an acclimation period prior to exposure. Animals were taken out of the chambers after completion of the 6.5-h protocol and placed in their home cages, except on day 0 or 3 (1- and 4-day exposure duration, respectively), at which time animals were sacrificed. Chambers were then washed, dried, and sprayed with the nonstatic solution in preparation for the next day.
Recordings of every 10 breaths were collected, extrapolated, and averaged at 1-min intervals using the BioSystem XA software (Buxco Electronics, Inc). Automated breath-by-breath analyses were performed using a rejection algorithm to eliminate breaths outside a given range. Data were averaged for each rat over each 6-h HP12 exposure period. For the 4-day HP12 exposure, parameters for individual animals were averaged across the 4 days.
Lung collection.
After the last exposure, rats were weighed, anesthetized with sodium pentobarbital (50–100 mg/kg, ip; Abbott Laboratories, Chicago, IL), and exsanguinated via the abdominal aorta. Rats exposed to HP12 were sacrificed prior to the air control rats to minimize the time available for particle clearance. The trachea, left lung lobe, right apical lobe, right intercostal lobe, right diaphragmatic lobe, and right cardiac lobe were individually dissected and cleared of blood and connective tissue. Each piece was separately weighed, placed in a sterile, labeled plastic vial, and stored on dry ice. After the tracheas and lung lobes had been collected for all rats, vials were stored at – 80°C.
NAA and deposition calculations.
All trachea and lung lobe samples were analyzed for Co, Mn, and V using NAA, as (1) HP12 has quantities of these trace elements in excess of the threshold for detection and (2) only minimal levels of these elements are found naturally in the rat body. Tissue samples (76–365 mg) were weighed and packaged in polyethylene vials, then irradiated in a PULSTAR nuclear reactor (North Carolina State University) according to element-specific protocols; the neutron flux for all samples was 1012 neutron/(cm2 · sec). For V and Mn content, samples were irradiated for 30 s, allowed to decay for 5 min, and subsequently counted for 5 min. For Co content, samples were irradiated for 18 h, allowed to decay for 5 weeks, then transferred to clean vials and counted for 15 min. Gamma ray spectra were counted on Ge(Li) detectors coupled to PC-based gamma spectroscopy systems (Omnigam-N 918A and 919; Ortec, Oak Ridge, TN). The detection limits were calculated based on statistical characteristics of the spectral photopeak signal-to-noise ratio for each element and ranged from 0.0084 to 0.012 µg/g for Co, from 0.085 to 0.24 µg/g for Mn, and from 0.012 to 0.099 µg/g for V.
The results from the NAA indicators were used to determine particle mass for HP12 dose calculations. The amounts of Co, Mn, and V in HP12 were established by subtracting the water-soluble portion of the trace element (as determined by inductively coupled plasma optical emission spectrometry) from the total trace element amount (as determined by x-ray fluorescence); this yielded the nonsoluble portion of trace element contained within HP12. Once this was completed, the following equation was used to calculate the measured dose for the trachea and lung lobes of each rat based on each element:
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Dose estimation.
The MPPD modeling software (v1.1; CIIT/RIVM; Anjilvel and Asgharian, 1995; Cassee et al., 1999) was used to predict deposition, retention, and clearance of HP12 for each rat. Model output included deposition for the following regions: (1) nasopharyngeal; (2) tracheobronchial; and (3) pulmonary. Additionally, to compare the predicted lower respiratory tract (LRT) deposition to the actual measured values, the MPPD model was used to calculate the deposited fraction of HP12 for separate lung lobes. Upper respiratory tract (URT) volume was calculated based on an allometric scaling equation using body weight (Ménache et al., 1997) and ranged from 0.36 to 0.40 ml. Similarly, functional residual capacity (FRC) was estimated as 0.5 of total lung capacity (TLC) for an unanesthetized rat (Overton et al., 1987), with TLC obtained from the equation provided by Takezawa et al. (1980). The FRC values input into the MPPD model ranged from 5.05 to 5.52 ml. Rat-specific respiration data obtained from the WBP were input into the model; these parameters included f, VT, and IF. Mucus velocity for rats was assumed to be 1.9 ml/min (Felicetti et al., 1981). The density of HP12 was assumed to be 1.0 g/cm3. For comparative purposes, the model was also run using default values for URT volume (0.42 ml), FRC (4.0 ml), f (102 breaths/min), VT (2.1 ml), and IF (0.5) as inputs. For all model runs, the inhalability adjustment was selected, and the pause fraction was the default value (0.0).
HP12 doses were estimated for each lung lobe using MPPD model predictions obtained from the following equation:
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Statistical methods.
For statistical analyses, deposition data were averaged across exposure times and distinguished as experimentally measured or predicted. Body and lung lobe weights were analyzed (SAS, Cary, NC) using one-way ANOVA to assess differences between sacrifice time and exposure group. Preplanned pairwise comparisons were made as subtests of the overall ANOVA only when the ANOVA was significant (p 
0.05), as determined using Fisher's least significant difference test (p 0.05).
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RESULTS |
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Exposure Concentrations
Target HP12 concentration was 13 mg/m3 for phases I and II. For the 1-day exposure study (phase I), the average concentration over 6 h was 13.1 mg/m3. Overall average HP12 concentration for the 4-day exposure study (phase II) was 13.2 mg/m3, with mean concentrations for each day of 13.2, 13.0, 13.3, and 13.1 mg/m3 for days 0, 1, 2, and 3, respectively.
Body and Lung Lobe Weights
Comparisons of body and lung lobe weights were made between the groups of animals sacrificed after the 1-day and the 4-day exposures to HP12 (Table 2). These data were further evaluated when lung lobes were normalized to body weight, as the average body weights differed between groups.
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At sacrifice, average body weights were approximately 16 g greater in rats exposed to HP12 for 4 days (270 ± 3.5 g) when compared to those of rats exposed for 1 day (254 ± 1.7 g). This was likely due to normal growth patterns for juvenile rats over the ensuing 4 days of exposure and is consistent with breeder growth charts. The left, right intercostal, and right diaphragmatic lung lobes weighed significantly more in the phase II rats compared to the phase I rats (Table 1). However, when these lobe weights were normalized to body weight (Table 3), there were no statistically significant differences between phase I and II rats.
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Deposition Calculations
As noted above, NAA was conducted for Co, Mn, and V on the tracheas and lung lobes of rats exposed to both air and HP12. Of these elements, concentrations of V were the most reliable, as this element yielded the most null values for control rats (i.e., values below the detection threshold); 23 of 24 measurements for the control rats (two rats per study x six lung tissues per rat x two studies) were below the detection limit. Concentrations of Co were present in 12 of 24 lung samples collected from rats exposed to air. In 11 of 12 tissues which had measurable concentrations of Co, all values were below the levels observed in HP12-exposed rats. In contrast, Mn had the greatest number of detectible values for air-exposed rats, some of which were higher than those for rats exposed to HP12. Because of the inherent variability of Mn in rat lungs, Mn values were not used in calculating HP12 dose. As HP12 doses obtained from V data were the most credible, they were employed for comparisons to MPPD model predictions, whereas Co results provided corroboration for the V findings. Measured V concentrations were lowest in the tracheas (Fig. 1a) for 1- and 4-day exposures. In contrast, average Co values for tracheas following a 1-day exposure to HP12 were similar to those observed in the right cardiac lobe (Fig. 1b). The V and Co concentrations for lung lobes collected after the 4-day exposure to HP12 were three to four times greater than those measured after a 1-day exposure (Figs. 1a and 1b), and the measured individual Co concentrations in lung tissues were much less than those observed with V.
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When V and Co concentrations were used to estimate the mass of HP12 deposited in the respiratory tract, the calculated HP12 dose was greater for most tissues with V (Fig. 2). The greatest consistent differences in HP12 deposition were observed in the trachea where, conversely, calculations using the Co data resulted in higher depositions for both 1- and 4-day exposures compared to V. Interestingly, the deposition for the 1-day exposure for the right cardiac lobe calculated from Co was less than that obtained using V; however, at 4 days the deposition was greater for Co than for that calculated with V.
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Despite these noted differences, there was good agreement between the total deposited doses calculated using V and Co for individual rats (R2 = 0.94; Fig. 3). Phase I and II averages of total HP12 deposition showed a similar trend to those of the individual tissues, with the calculations using V resulting in higher HP12 doses compared to those obtained using Co. Total deposition was calculated as 26.2 ± 6.4 µg and 99.2 ± 9.2 µg for Co at 1 and 4 days, respectively; for V at 1 and 4 days, HP12 dose was calculated as 31.0 ± 6.2 µg and 116 ± 6.9 µg, respectively.
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WBP Parameters
When possible, rat-specific breathing parameters were incorporated in the MPPD model runs; these data included average f, VT, and IF (Table 4). For rats exposed to HP12 for 1 day (phase I), average f and VT were slightly higher on day–1 when compared to day 0; whereas, average IF was slightly increased in five of six rats on day 0 compared to day–1. For 4-day exposures (phase II), similar trends held for average f and IF, while average VT was increased over 4 days compared to day–1. One rat (rat 14) had consistently greater f and lower VT than the other rats, with the former ranging from 98 to 110 breaths/min over the experimental period.
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MPPD Model Runs
Runs using the MPPD modeling software at default settings resulted in larger estimates of HP12 pulmonary deposition (without consideration of particle clearance) for all lung lobes compared to those obtained when the model was used with rat-specific breathing parameters for both 1- and 4-day exposures (Figs. 4a and 4b). These results highlight the importance of obtaining respiratory physiology parameters that can be input into the MPPD model for dose predictions.
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As shown in Figure 5, the MPPD model underestimated HP12 deposition (with rat-specific inputs) compared to experimentally obtained doses. The measured deposition exceeded the model prediction by a factor of > 3. Total particle clearance (defined as mucociliary transport from the tracheobronchial region and translocation to lymph nodes from the alveolar region) as calculated by the MPPD model was estimated to be 4.4 (± 0.3) and 41 (± 2) µg for the 1- and 4-day exposures, respectively. Particle clearance was not empirically determined in this study, rather it is estimated from dose differences between the 1- and 4-day exposures.
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Lung Deposition Fractions of HP12
The MPPD model also provided deposition estimates for the fraction of HP12 aerosol that deposited in the respiratory tract, based on rat-specific inputs. For the rats exposed to HP12 during phase I, the average predicted total deposition fraction in the LRT was 2.8 ± 0.2%; separation of this deposition fraction into its tracheobronchial and alveolar deposition fraction components yielded values of 1.2 ± 0.1% and 1.6 ± 0.2%, respectively. These values were roughly the same for the 4-day HP12 exposure, with total deposition fraction equal to 2.7 ± 0.2%, tracheobronchial deposition equal to 1.2 ± 0.0%, and alveolar deposition equal to 1.5 ± 0.1%. The model also estimated nasal deposition as 25 and 26% of the exposure concentration for the 1- and 4-day exposures, respectively.
Deposition percentages by lung lobe are shown in Figures 6a and 6b for measured and estimated HP12 dose. There is little difference between the measured and predicted distribution values for 1- and 4-day exposures to HP12; the greatest differential was observed for the left lung lobe. There were two lung lobes in which the model predicted a greater percentage deposition than that measured, namely, the right diaphragmatic and right cardiac lobes. It should be noted that the MPPD model does not estimate tracheal deposition.
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The MPPD model also provided deposition calculations per region (i.e., conducting airways and alveolar) for each lung lobe. Similar to the estimates of deposition percentage by lung lobe, there were no differences between regional depositions after 1- and 4-day exposures to HP12 (Fig. 7). Thus, for each lung lobe at each time point, an average of 43% of HP12 was estimated to deposit in the conducting airways, with the remaining 57% deposited in the alveolar regions. Although the distribution of HP12 across the conducting airways and alveolar regions was not determined experimentally in this study, it is probable that the model predictions are a close approximation of the actual deposition, as the measured deposition percentages per lung lobe (Figs. 6a and 6b) were reasonably close to the model predictions.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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We have developed a rodent whole-body exposure system for PM that permits continuous noninvasive measurement of pulmonary ventilation parameters throughout the exposure period (Wichers et al., in press). In earlier animal inhalation studies investigating PM, minute ventilation (VE) was not acquired during exposure, but rather was estimated or assumed as some default value (Campen et al., 2001
; Kodavanti et al., 2003). For the present study, however, rat breathing parameters were measured during exposure to PM, and the resultant data were used as input variables for the MPPD model in order to better estimate dose. The values thus obtained were then compared to actual lung compartment (lobar) doses to assess disparities in the deposition data.
In a recent study similar to the present study, ultrafine particles and nose-only inhalation exposures were used to assess deposition and to provide comparisons to MPPD modeling results (Cassee et al., 2002). In that study, it was reported that pulmonary deposition of soluble CdCl2 particles in rats after a single 4-h exposure to either 0.63 mg/m3 (count median diameter = 1.5 µm) or 1.43 mg/m3 (count median diameter = 0.033 µm) was 0.1 or 3.0 µg, respectively (Cassee et al., 2002). Similar to the results of the current study, the highest particle doses were seen in the left lung and the right diaphragmatic lobes, with the remaining lobes having approximately the same depositions. The MPPD model prediction with the CdCl2 particles overestimated measured pulmonary deposition when f and VT were input as rat-specific parameters (Cassee et al., 2002), as only 15% of the predicted dose was recovered in lung tissue. Part of this discrepancy is likely due to the high solubility of the CdCl2 particles. The MPPD model was much more accurate in predicting lung dose for the current study, albeit model predictions were < 33% of the measured dose.
The MPPD model consistently underpredicted the mass of HP12 deposited in the lung lobes; there are several possible explanations for these discrepancies. First, the large GSD of HP12 likely impacted deposition and may have resulted in inaccurate MPPD model outputs. Another possibility is that a greater amount of HP12 deposited in the distal airways compared to that approximated by the MPPD model. The MPPD model could have also underestimated the mass of HP12 inhaled from the exposure chamber. Finally, MPPD model overestimation of particle deposition in the nasal region could explain the discrepancies between model predictions and experimentally obtained data.
In another study, Costa and colleagues (2006) conducted a nose-only exposure to 12 mg/m3 of residual oil fly ash (MMAD = 1.95 µm) in rats for the same duration as the present study, resulting in depositions in the left lung of 32 µg and in the right diaphragmatic lobe of 29 µg; these findings are at least three times the deposition observed after a whole-body inhalation exposure with nearly the same experimental conditions (e.g., exposure duration, concentration, and particle size). While pulmonary function was not measured in the Costa et al. (2006) nose-only study, minute ventilation was obtained in the Cassee et al. (2002) study and found to be approximately three times that observed in the current work. Therefore, it would appear that restraint of the rodents during exposure likely increased ventilation and hence, particle dose; similarly, as the rats in the current study tended to sleep during exposure, respiration was slowed and this change in responsiveness could partially explain differences in observed doses.
The MPPD model breathing scenario default values for rats are as follows: 102 breaths/min for f, 2.1 ml for VT, 0.5 s for IF, and 0 s for pause, values which were first measured in rats at rest (Mauderly et al., 1979). However, the use of default values in the model yielded dose values for HP12 closer to those observed compared to rat-specific inputs. In the present study, data collected on the durations of breathing cycles enabled calculation of IF. The TI results ( 0.25 s) were similar to those for Sprague-Dawley rats of 305 g (0.25 s), but greater than those for Wistar-Kyoto rats of 300 g (0.19 s) (reviewed in Boggs, 1992). However, when TI was calculated as a fraction of the total time of the respiratory cycle for Wistar-Kyoto rats, the ratio was 0.35–0.42. Similarly, the IF results for SH rats in the current study were between 0.34 and 0.37, well within the range of previously determined rat values. Interestingly, IF does not appear to vary much between species and, in comparisons with the IFs of 11 different mammals, the IF obtained in the present study (0.35) was the same as the average of these species (Boggs and Tenney, 1984). Thus, it appears that for an unanesthetized rodent exposed via whole-body inhalation, the appropriate IF value for input into the MPPD model is likely 0.35.
In the present study, the 1- and 4-day data show that the amount of HP12 remaining in the lungs following a 4-day exposure was 3.4 times that observed after a 1-day exposure. This was an unexpected finding, as it was hypothesized that clearance mechanisms would effectively reduce HP12 quantities in the lungs. Given the HP12 size fraction, bronchial clearance should have eliminated a large portion of deposited particles (Hofmann and Asgharian, 2003; Snipes et al., 1983), particularly given the 18-h periods between exposures. However, this did not appear to be the case. The observed HP12 accumulation is likely not attributable to lung overload, as this condition is characterized by (1) > 1 mg of accumulated PM (Morrow, 1988; Oberdorster, 1995), (2) long-term exposure, and (3) poorly soluble particles.
Another possible explanation for the HP12 doses observed following the 4-day exposure is rat strain. It has been previously shown that Fischer 344 rats retain more TiO2 (MMAD = 1.0) particles than Long-Evans rats following a 7-h whole-body inhalation (Ferin and Morehouse, 1980). As the MPPD model is based on the lung morphometry of a Long-Evans rat and the SH rat was employed in this work, the discrepancy between the measured and MPPD predicted doses in the present study may be partially attributable to strain.
The effect of reduced particle clearance may have been reflected in the amount of HP12 measured in the trachea after the 1- and 4-day inhalation exposures. The average levels of HP12 present in the trachea after 1 day were double those observed after the 4-day exposure (0.52 and 0.26 µg, respectively). Thus, a 1-day HP12 exposure may have induced mild irritation in the airways, while the initial deposition of particles stimulated mucosal production, both factors which could promote clearance in the trachea (Schlesinger, 1995; Wolff, 1992). With higher concentrations of aerosols (or in this case, repeated exposure), mucus velocities have been shown to dramatically decrease and impair clearance (Schlesinger, 1995; Wolff, 1992), which could contribute to the decrease in particle mass in the trachea observed in the present study after the 4-day exposure to HP12. However, this is purely speculation as no measurements of clearance were made during this study.
Interestingly, rat 6 had a higher deposition than any other rat exposed to HP12 for 1 day, in the absence of differences in breathing parameters (f = 91.2 breaths/min; VT = 1.3 ml; IF = 0.35); total deposition for this rat was calculated to be between 54 and 60 µg (based on Co and V concentrations, respectively), a value more than double that observed with other rats in this group. This elevated deposition may have been attributable to differences in lung morphometry or specific URT characteristics of rat 6 that resulted in more HP12 penetration. While it might be tempting to exclude this rat from the mean calculations, as it could be considered an "outlier" compared to the rest of the exposure animals, in fact, this rat may represent those in the population that are more vulnerable (based on dosimetry) to PM toxicological effects. Given this possibility, perhaps greater focus should be placed on the data furthest from the average for the evaluation of susceptible individuals.
Regional deposition, as predicted by the MPPD model for the rats in this study, was roughly 40% in the tracheobronchial region (i.e., conducting airways) and 60% in the alveolar region. Surface areas (SAs) of these regions in the rat have been reported as 23.46 and 2970 cm2, respectively (Yeh and Schum, 1979), resulting in a ratio of airway SA:alveolar SA of approximately 1:127. If these regional deposition percentages are displayed in terms of SA per lung lobe, it becomes apparent that there is 1000 times the deposition per SA in the tracheobronchial region as there is in the alveolar region (Table 5). This could have significant implications in assessing PM-induced modes of action, as pathways of neurological and inflammatory origin have both been suggested as mediators of the cardiopulmonary effects resulting from exposure to particles.
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It is possible to extrapolate these approximations of HP12 deposition per SA unit for the tracheobronchial and alveolar regions to humans. Recently, equations have been presented by Brown et al. (2005)
and Jarabek et al. (2005) which provide the means to calculate human equivalent exposure concentrations (HEECs) that would result in roughly the same deposition per normalizing factor, such as SA or macrophage number. Values for different HEECs are provided in Table 6, which were calculated for human exposure scenarios (e.g., rest and light, moderate, and heavy exercise; oronasal normal augmenter and oronasal mouth breather) using average rat data measured in the current study and obtained under the same conditions (i.e., one 6-h exposure duration and HP12 MMAD = 1.95 µm and g = 3.49). Humans have a greater particle burden in the tracheobronchial region compared to that of the rat and also have slower clearance rates from the alveolar region (Brown et al., 2005; U.S. EPA, 1996). Moreover, the large g associated with the HP12 size distribution makes interpretation of these equivalent concentrations difficult due to uncertainties with particle deposition and clearance. Despite these caveats, this attempt to extrapolate these rat deposition results to humans represents a significant step forward that should aid risk assessors in relating exposure to toxicological outcome.
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The above research demonstrates that rodent whole-body inhalation of a polydisperse PM results in much lower pulmonary deposition than that reported previously with nose-only exposures. Additionally, the quantity of HP12 in the lung lobes following a 4-day exposure to 13 mg/m3 is three to four times that observed after a 1-day exposure under the same experimental conditions. When measured particle deposition is compared to MPPD model estimations, the predictions are closest to actual values when default inputs are used. Finally, extrapolation of these findings to a human exposure scenario that would result in roughly the same deposited dose provides important linkages between the effects of PM observed in animal studies and the impact of PM on human health.
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NOTES |
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Disclaimer: This paper has been reviewed by the National Health and Environmental Effects Research Laboratory and the National Center for Environmental Assessment, U.S. Environmental Protection Agency, and approved for publication. Mention of trade names or commercial products does not constitute endorsement or recommendation for use.
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ACKNOWLEDGMENTS |
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The authors acknowledge Drs Linda S. Birnbaum, James S. Brown, Urmila P. Kodavanti, and Michael C. Madden for their helpful discussions and thoughtful reviews of the manuscript. The authors also acknowledge Dr John J. Vandenberg for contributing to the scope of this project and Jerry W. Highfill for offering statistical advice. A. Glenn Ross and John A. Sullivan (The National Caucus and Center on Black Aged Inc./Senior Environmental Employment Program, Research Triangle Park, NC) and Scott A. Lassell (Department of Nuclear Engineering, North Carolina State University) provided excellent analytical support for lung lobe preparation and NAA. Dr Urmila P. Kodavanti and Mette C. J. Schladweiler also provided technical support. Drs Russ Hauser and David Christiani of the Harvard School of Public Health supplied the HP12 bulk sample through grant ES00002. This work was partially supported through a cooperative training agreement (EPA/UNC CT826513) with the University of North Carolina at Chapel Hill.
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REFERENCES |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Anjilvel, S., and Asgharian, B. (1995). A multiple-path model of particle deposition in the rat lung. Fundam. Appl. Toxicol. 28, 41–50.[CrossRef][ISI][Medline]
Asgharian, B., Kelly, J. T., and Tewksbury, E. W. (2003). Respiratory deposition and inhalability of monodisperse aerosols in Long-Evans rats. Toxicol. Sci. 71, 104–111.
Boggs, D. F. (1992). Comparative control of respiration. In Treatise on Pulmonary Toxicology, Volume I: Comparative Biology of the Normal Lung (R. A. Parent, Ed.), Vol. 1, pp. 217–239. CRC Press, Boca Raton, FL.
Boggs, D. F., and Tenney, S. M. (1984). Scaling respiratory pattern and respiratory ‘drive’. Respir. Physiol. 58, 245–251.[CrossRef][ISI][Medline]
Brook, R. D., Brook, J. R., and Rajagopalan, S. (2003). Air pollution: the "Heart" of the problem. Curr. Hypertens. Rep. 5, 32–39.[ISI][Medline]
Brown, J. S., Wilson, W. E., and Grant, L. D. (2005). Dosimetric comparisons of particle deposition and retention in rats and humans. Inhal. Toxicol. 17, 355–385.[ISI][Medline]
Brunekreef, B., and Holgate, S. T. (2002). Air pollution and health. Lancet 360, 1233–1242.[CrossRef][ISI][Medline]
Campen, M. J., Nolan, J. P., Schladweiler, M. C., Kodavanti, U. P., Evansky, P. A., Costa, D. L., and Watkinson, W. P. (2001). Cardiovascular and thermoregulatory effects of inhaled PM-associated transition metals: A potential interaction between nickel and vanadium sulfate. Toxicol. Sci. 64, 243–252.
Cassee, F., Freijer, J., Subramaniam, R., Asgharian, B., Miller, F., van Bree, L., and Rombout, P. (1999). Development of a Model for Human and Rat Airway Particle Deposition: Implications for Risk Assessment (DNIPHE [RIVM], Ed). RIVM, Bilthoven, Netherlands.
Cassee, F. R., Muijser, H., Duistermaat, E., Freijer, J. J., Geerse, K. B., Marijnissen, J. C., and Arts, J. H. (2002). Particle size-dependent total mass deposition in lungs determines inhalation toxicity of cadmium chloride aerosols in rats. Application of a multiple path dosimetry model. Arch. Toxicol. 76, 277–286.[CrossRef][ISI][Medline]
Chen, B. T., Weber, R. E., Yeh, H. C., Lundgren, D. L., Snipes, M. B., and Mauderly, J. L. (1989). Deposition of cigarette smoke particles in the rat. Fundam. Appl. Toxicol. 13, 429–438.[CrossRef][ISI][Medline]
Costa, D. L., Lehmann, J. R., Winsett, D., Richards, J., Ledbetter, A. D., and Dreher, K. L. (2006). Comparative pulmonary toxicological assessment of oil combustion particles following inhalation or instillation exposure. Toxicol. Sci. 91, 237–246.
de Winter-Sorkina, R., and Cassee, F. R. (2002). From Concentration to Dose: Factors Influencing Airborne Particulate Matter Deposition in Humans and Rats (DNIPHE [RIVM] Ed). RIVM, Bilthoven, Netherlands.
Dorato, M. A., and Wolff, R. K. (1991). Inhalation exposure technology, dosimetry, and regulatory issues. Toxicol. Pathol. 19, 373–383.[ISI][Medline]
Driscoll, K. E., Costa, D. L., Hatch, G., Henderson, R., Oberdorster, G., Salem, H., and Schlesinger, R. B. (2000). Intratracheal instillation as an exposure technique for the evaluation of respiratory tract toxicity: Uses and limitations. Toxicol. Sci. 55, 24–35.
Felicetti, S. A., Wolff, R. K., and Muggenburg, B. A. (1981). Comparison of tracheal mucous transport in rats, guinea pigs, rabbits, and dogs. J. Appl. Physiol. 51, 1612–1617.
Ferin, J., and Morehouse, B. (1980). Lung clearance of particles in two strains of rats. Exp. Lung Res. 1, 251–257.[ISI][Medline]
Hofmann, W., and Asgharian, B. (2003). The effect of lung structure on mucociliary clearance and particle retention in human and rat lungs. Toxicol. Sci. 73, 448–456.
Jarabek, A. M., Asgharian, B., and Miller, F. J. (2005). Dosimetric adjustments for interspecies extrapolation of inhaled poorly soluble particles. Inhal. Toxicol. 17, 317–334.[ISI][Medline]
Kodavanti, U. P., Moyer, C. F., Ledbetter, A. D., Schladweiler, M. C., Costa, D. L., Hauser, R., Christiani, D. C., and Nyska, A. (2003). Inhaled environmental combustion particles cause myocardial injury in the Wistar Kyoto rat. Toxicol. Sci. 71, 237–245.
Ledbetter, A. D., Killough, P. M., and Hudson, G. F. (1998). A low-sample-consumption dry-particulate aerosol generator for use in nose-only inhalation exposures. Inhal. Toxicol. 10, 239–251.[CrossRef]
Mauderly, J. L., Tesarek, J. E., and Sifford, L. J. (1979). Respiratory measurements of unsedated small laboratory mammals using nonrebreathing valves. Lab. Anim. Sci. 29, 323–329.[ISI][Medline]
Menache, M. G., Hanna, L. M., Gross, E. A., Lou, S. R., Zinreich, S. J., Leopold, D. A., Jarabek, A. M., and Miller, F. J. (1997). Upper respiratory tract surface areas and volumes of laboratory animals and humans: considerations for dosimetry models. J. Toxicol. Environ. Health 50, 475–506.[CrossRef][ISI][Medline]
Morrow, P. E. (1988). Possible mechanisms to explain dust overloading of the lungs. Fundam. Appl. Toxicol. 10, 369–384.[CrossRef][ISI][Medline]
Newton, P. E., and Pfledderer, C. (1986). Measurement of the deposition and clearance of inhaled radiolabeled particles from rat lungs. J. Appl. Toxicol. 6, 113–119.[ISI][Medline]
Oberdorster, G. (1995). Lung particle overload: Implications for occupational exposures to particles. Regul. Toxicol. Pharmacol. 21, 123–135.[CrossRef][ISI][Medline]
Overton, J. H., Graham, R. C., and Miller, F. J. (1987). A model of the regional uptake of gaseous pollutants in the lung. II. The sensitivity of ozone uptake in laboratory animal lungs to anatomical and ventilatory parameters. Toxicol. Appl. Pharmacol. 88, 418–432.[CrossRef][ISI][Medline]
Schlesinger, R. B. (1995). Toxicological evidence for health effects from inhaled particulate pollution: Does it support the human experience? Inhal. Toxicol. 7, 99–109.
Snipes, M. B., Boecker, B. B., and McClellan, R. O. (1983). Retention of monodisperse or polydisperse aluminosilicate particles inhaled by dogs, rats, and mice. Toxicol. Appl. Pharmacol. 69, 345–362.[CrossRef][ISI][Medline]
Snipes, M. B., Olson, T. R., and Yeh, H. C. (1988). Deposition and retention patterns for 3-, 9-, and 15-micron latex microspheres inhaled by rats and guinea pigs. Exp. Lung Res. 14, 37–50.[ISI][Medline]
Takezawa, J., Miller, F. J., and O'Neil, J. J. (1980). Single-breath diffusing capacity and lung volumes in small laboratory mammals. J. Appl. Physiol. 48, 1052–1059.
U.S. EPA (1996). Air Quality Criteria for Particulate Matter, Vol. I–III, pp. 163–169. Office of Research and Development, National Center for Environmental Assessment, Research Triangle Park, NC.
U. S. EPA (2004). Air Quality Criteria for Particulate Matter, Vol. I–III. U.S. EPA, Office of Research and Development, National Center for Environmental Assessment, Research Triangle Park, NC.
Watkinson, W. P., Campen, M. J., Nolan, J. P., and Costa, D. L. (2001). Cardiovascular and systemic responses to inhaled pollutants in rodents: effects of ozone and particulate matter. Environ. Health Perspect. 109(Suppl. 4), 539–546.[ISI][Medline]
Wichers, L. B., Ledbetter, A. D., McGee, J. K., Kellogg, R. B., Rowan, W. H., Nolan, J. P., Costa, D. L., and Watkinson, W. P. (2006). A method for exposing rodents to resuspended particles using whole-body plethysmography. Part Fibre Toxicol. Available at: http://www.particleandfibretoxicology.com/content/pdf/1743-8977-3-12.pdf. Accessed August 15, 2006.
Wichers, L. B., Nolan, J. P., Winsett, D. W., Ledbetter, A. D., Kodavanti, U. P., Schladweiler, M. C., Costa, D. L., and Watkinson, W. P. (2004a). Effects of instilled combustion-derived particles in spontaneously hypertensive rats. Part I: Cardiovascular responses. Inhal. Toxicol. 16, 391–405.[CrossRef][ISI][Medline]
Wichers, L. B., Nolan, J. P., Winsett, D. W., Ledbetter, A. D., Kodavanti, U. P., Schladweiler, M. C., Costa, D. L., and Watkinson, W. P. (2004b). Effects of instilled combustion-derived particles in spontaneously hypertensive rats. Part II: Pulmonary responses. Inhal. Toxicol. 16, 407–419.[CrossRef][ISI][Medline]
Wolff, R. K. (1992). Mucociliary function. In Treatise on Pulmonary Toxicology, Volume I: Comparative Biology of the Normal Lung (R. A. Parent, Ed.), Vol. I, pp. 659–680. CRC Press, Boca Raton, FL.
Yeh, H. C., and Schum, G. M. (1979). Anatomic models of the tracheobronchial and pulmonary regions of the rat. Anat. Rec. 195, 483–492.[CrossRef][Medline]
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