In some cases, the surface coatings had little or no impact Pulmonary bioassay bridging study Inhalation studies Carbonyl iron particles α-quartz particles Intratracheal instillation stu
Trang 118 Models for Testing the
Pulmonary Toxicity of Particles: Lung Bioassay Screening Studies
in Male Rats with a New
David B Warheit, Kenneth L Reed, and Christie M Sayes DuPont Haskell Laboratory for Health and Environmental Sciences
CONTENTS
18.1 Introduction 317
18.2 Methods 319
18.2.1 General Experimental Design 319
18.2.2 Animals 320
18.2.3 Particle Types 320
18.2.4 Lung Cell Proliferation Studies 320
18.2.5 Bronchoalveolar Lavage Methods 321
18.2.6 Lung Histopathology Studies 321
18.2.7 Statistical Analyses 321
18.3 Results 321
18.3.1 Lung Weights 321
18.3.2 Lung Cell Proliferation Results 321
18.3.3 Bronchoalveolar Lavage Fluid Results 323
18.3.3.1 Pulmonary Inflammation 323
18.3.3.2 Bronchoalveolar Lavage (BAL) Fluid Parameters 323
18.3.3.3 Pulmonary Histopathological Evaluations 325
18.4 Discussion 325
Acknowledgments 329
References 329
18.1 INTRODUCTION
Formulation changes in the form of altered surface treatments are known to frequently occur for a variety of commercialized particle-types The R-100 formulation of rutile-type titanium dioxide is a well-known low-toxicity particulate This study was designed as a pulmonary screening tool to
317
Trang 2encapsulated with pyrogenically deposited amorphous silica) may impart significant toxicity in the
study was to evaluate in the lungs of rats—using a well-developed, short-term pulmonary
samples and to compare the pulmonary toxicity of these samples with two other low toxicity particulate-types (reference negative controls), along with a cytotoxic particulate (reference positive control) sample Another aim was to bridge the results obtained herein with data previously generated from inhalation studies with crystalline silica-quartz particles and with carbonyl iron particulates as the inhalation/instillation bridge materials
Bridging studies can be useful in providing an inexpensive safety screen when assessing the hazards of new developmental compounds or when making small modifications to an existing commercial particle-type, such as surface treatments The strength of the bridging strategy is dependent upon having good inhalation toxicity data on one of the bridging compounds The particle-type for which inhalation data exists can then be used as a reference control material for
an intratracheal instillation bridging study (see Figure 18.1) The basic idea for the bridging concept
is that the effects of the instilled material serve as a control (known reference) material and then are
“bridged” on the one hand to the inhalation toxicity data for that material, as well as to the new materials being tested The results of bridging studies in rats are then useful as pulmonary toxicity screening (i.e., hazard) data, because consistency in the response of the inhaled and instilled control material serves to validate the responses with the newly tested particulate matter
Numerous studies have investigated the pulmonary toxicological impacts of surfaces treat-ments on titanium dioxide particles In some cases, the surface coatings had little or no impact
Pulmonary bioassay bridging study
Inhalation studies
Carbonyl iron particles
α-quartz particles
Intratracheal instillation studies
PBS sham α-quartzparticles
Carbonyl iron particles
R-100 &
Pigment A TiO2 particles
FIGURE 18.1 Schematic demonstrating the strategy for conducting pulmonary bioassay bridging studies Bridging studies can have utility in providing an inexpensive preliminary safety screen when evaluating the hazards of new developmental compounds The basic idea for the bridging concept is that the effects of the instilled material serve as a control (known) material and are then “bridged” to the inhalation toxicity data for that material and to the new materials being tested
Trang 3current study describes a model or pulmonary bioassay method for assessing the pulmonary hazard potential of intratracheally instilled particle-types The chapter also discusses the relevance of this screening methodology as a possible surrogate for inhalation studies with low-solubility particle-types
18.2 METHODS
18.2.1 GENERALEXPERIMENTALDESIGN
The fundamental features of this pulmonary bioassay are dose-response evaluation, time-course assessments, and reference particle-types (positive and negative) The time-course studies are used
to assess the sustainability of the observed affect The major endpoints of this study were the (1) time-course and dose/response intensity of pulmonary inflammation and cytotoxicity (bronchoal-veolar lavage (BAL) parameters), (2) airway and lung parenchymal cell proliferation, and (3) histopathological evaluation of lung tissue (see Figure 18.2)
The lungs of rats were exposed via intratracheal instillation with single doses of 1 or 5 mg/kg
of entry technique is not a substitute for the more physiologically relevant inhalation method of exposure However, the intratracheal instillation method of exposure can be a qualitatively reliable screen for assessing the pulmonary toxicity of particles [1,2] All particles were prepared in a volume of phosphate-buffered saline (PBS) solution and subjected to probe sonication for at least 15 minutes Groups of PBS-instilled rats served as controls The lungs of PBS and particle-exposed rats were evaluated by BAL fluid analyses at 24 h, 1 week, 1 month, and 3 months postexposure (pe) For lung cell proliferation and histopathology studies, additional groups of animals were instilled with the particle-types listed above as well as a PBS solution
For the lung tissue studies, additional groups of animals (4 rats/group) were instilled with the particle-types listed above plus the vehicle control, i.e., PBS These studies were dedicated
to lung tissue analyses, but only the high-dose groups (5 mg/kg) and PBS controls were utilized
in the morphology studies These studies consisted of cell proliferation assessments and histo-pathological evaluations of the lower respiratory tract Similar to the BAL fluid studies, the intratracheal instillation exposure period was followed by 24-h, 1-week, 1-month, and 3-month recovery periods
Exposure groups
•
• PBS (vehicle control)Particle-types (1 and 5 mg/kg)
o R-100 fine-TiO2
o Pigment A-fine-TiO2coated with amorphous SiO2
o α-Quartz particles (positive control)
o Carbonyl iron (negative control) Instillation
exposure
Postexposure (pe) evaluation via BAL and lung tissue
24 h 1 week 1 month 3 months
FIGURE 18.2 Experimental design for bridging case study
Trang 418.2.2 ANIMALS
Carolina) were used in this study The rats were approximately 8 weeks old at study start (mean weights in the range of 240–255 g) All procedures using animals were reviewed and approved by the Institutional Animal Care and Use Committee and the animal program is fully accredited by the Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC)
18.2.3 PARTICLETYPES
The R-100 fine-titanium dioxide particles (w99 wt.% titanium dioxide, w1 wt.% alumina)
obtained from the DuPont Company A patented chloride process produced Pigment A
dioxide, w1 wt.% alumina, w3 wt.% amorphous silica (particle encapsulating)) possessing an
in the rutile crystal phase and hydrophilic in nature
Crystalline silica particles (a-quartz, Min-U-Sil 5) ranging in size from 0.3 to 3 mm were obtained from the US Silica Company Carbonyl iron (CI) particles ranging in size from 0.8 to 3.0 mm were obtained from GAF Corporation
18.2.4 LUNGCELLPROLIFERATIONSTUDIES
Groups of particulate-exposed rats and corresponding controls were pulsed 24 h after instillation,
as well as 1 week, 1 month, and 3 months postexposure, with an intraperitoneal injection of
dose of 100 mg/kg body weight The animals were euthanized 6 h later by pentobarbital injection Following cessation of spontaneous respiration, the lungs were infused with a neutral buffered
and the heart and lungs were carefully removed en bloc and immersion-fixed in formalin In addition, a 1 cm piece of duodenum (which served as a positive control) was removed and stored in formaldehyde Subsequently, parasagittal sections from the right cranial and caudal lobes and regions of the left lung lobes as well as the duodenal sections were dehydrated in 70% ethanol and sectioned for histology The sections were embedded in paraffin, cut, and mounted on glass slides The slides were stained with an anti-BrdU antibody, with an AEC (3-amino-9-ethyl carbazole) marker, and counter-stained with aqueous hematoxylin A minimum of 1000 cells/animal were counted in terminal bronchiolar and alveolar regions For each treatment group, immunostained nuclei in airways (i.e., terminal bronchiolar epithelial cells)
TABLE 18.1
and a-Quartz Particulates
Particle Crystallinity Surface Area (m 2 /g) Average ParticleDiameter (nm)
Pigment A coated with amorph SiO 2 Rutile 8 w290
Trang 5or lung parenchyma (i.e., epithelial, interstitial cells, or macrophages) were counted by light
18.2.5 BRONCHOALVEOLARLAVAGEMETHODS
The lungs of sham and particulate-exposed rats were lavaged with a warmed PBS solution as described previously Methodologies for cell counts, differentials, and pulmonary biomarkers in lavaged fluids were conducted as previously described [3,4] Briefly, the first 12 mL of lavaged fluids recovered from the lungs of PBS or particulate-exposed rats was centrifuged at 700 g, and
2 mL of the supernatant was removed for biochemical studies All biochemical assays were
alka-line phosphatase (ALP), and lavage fluid protein were measured using Roche Diagnostics
injury ALP activity is a measure of Type II alveolar epithelial cell secretory activity, and increased ALP activity in BAL fluids is considered to be an indicator of Type II lung epithelial cell toxicity Increases in BAL fluid micro protein (MTP) concentrations generally are consistent with enhanced permeability of vascular proteins into the alveolar regions, indicating a breakdown in the integrity
of the alveolar-capillary barrier
18.2.6 LUNGHISTOPATHOLOGYSTUDIES
The lungs of rats exposed to particulates or PBS controls were prepared for light microscopy by
month, and 3 months postexposure Sagittal sections of the left and right lungs were made using a razor blade Tissue blocks were dissected from left, right upper, and right lower regions of the lung and were subsequently prepared for light microscopy (paraffin embedded, sectioned, and hematox-ylin–eosin stained) [3,4]
18.2.7 STATISTICALANALYSES
For analyses, each of the experimental values were compared to their corresponding sham control values for each time point A one-way analysis of variance (ANOVA) and Bartlett’s test were calculated for each sampling time When the F test from ANOVA was determined to be significant, the Dunnett’s test was utilized to compare means from the control group and each of the groups exposed to particulates Significance was judged at the 0.05 probability level
18.3 RESULTS
18.3.1 LUNGWEIGHTS
Lung weights of rats were enhanced with increasing age on the study (i.e., increased postexposure time periods following intratracheal instillation exposures) Lung weights in high-dose quartz-exposed rats were slightly increased relative to controls at 1 week, and at 1 month and 3 months postexposure (data not shown)
18.3.2 LUNGCELLPROLIFERATIONRESULTS
Tracheobronchial cell proliferation rates (percentage of immunostained cells with BrdU) were measured only in high-dose (5 mg/kg) particulate-exposed rats and corresponding controls at
24 h, 1 week, 1 month, and 3 months postexposure (pe) Although increases in cell labeling
Trang 6indices were measured in R-100 fine-TiO2as well as a-quartz-exposed animals at 24 h postexpo-sure, these effects were not sustained (data not shown)
Lung parenchymal cell proliferation rates (percentage of immunostained cells with BrdU) were measured only in high-dose (5 mg/kg) particulate-exposed rats and corresponding controls at 24 h,
1 week, 1 month, and 3 months postexposure (pe) Small but significant transient increases in lung
particle-exposed rats at 24 h, but these effects were not sustained at any other postexposure time points Significantly larger increases in cell proliferation indices were measured in the lungs of a-quartz exposed rats measured from 24 h postexposure through 3 months postexposure (data not shown)
To summarize, exposures to 5 mg/kg quartz particles produced increased tracheobronchial cell proliferation compared to PBS controls, but increases were statistically significant only at 24 h postexposure In contrast to tracheobronchial cell labeling indices, exposures to 5 mg/kg quartz particles produced substantially greater lung parenchymal cell proliferation rates at all time points postexposure, suggesting a greater likelihood to result in lung cell genotoxicity or other adverse pulmonary effects over time with continued exposures
Total cells in BAL fluids of rats exposed to pigment A TiO2particles and other particulates
0.00E+00 5.00E+06 1.00E+07 1.50E+07 2.00E+07 2.50E+07 Control
1 mg/kg
5 mg/kg
1 mg/kg
5 mg/kg
1 mg/kg
5 mg/kg
1 mg/kg
5 mg/kg
Mean number total cells in BAL fluids
3 month
1 month
1 week
24 h
FIGURE 18.3 Total number of cells in BAL fluids recovered from particulate-exposed rats and controls as evidenced by % neutrophils (PMN) in BAL fluids at 24 h, 1 week, 1 month, and 3 months postexposure (pe) Values given are meansGS.D The numbers of BAL cells recovered from the lungs of high-dose quartz groups were substantially higher than any other groups for all postexposure time periods
Trang 718.3.3 BRONCHOALVEOLARLAVAGEFLUIDRESULTS
18.3.3.1 Pulmonary Inflammation
The numbers of cells recovered by BAL from the lungs of high-dose a-quartz-exposed (5 mg/kg) groups were substantially higher than any of the other groups for all postexposure time periods (Figure 18.3) Intratracheal instillation exposures of virtually all particle-types produced a short-term pulmonary inflammatory response, as evidenced by an increase in the percentages/numbers of BAL-recovered neutrophils, measured at 24 h postexposure However, only the exposures to a-quartz particles (1 and 5 mg/kg) produced sustained pulmonary inflammatory responses, as measured through 3 months postexposure (Figure 18.4)
18.3.3.2 Bronchoalveolar Lavage (BAL) Fluid Parameters
Transient and reversible increases in (BAL) fluid LDH values, as an indicator of general
week postexposure, but were not sustained through the other postexposure time periods In contrast, exposures to high-dose (5 mg/kg) a-quartz particles produced a persistent enhancement in BAL
Percent neutrophils in BAL fluids of rats exposed to pigment A TiO2particles and other particulates
Control
1 mg/kg
5 mg/kg
1 mg/kg
5 mg/kg
1 mg/kg
5 mg/kg
1 mg/kg
5 mg/kg
Carbonyl iron
Mean % PMNs
3 month
1 month
1 week
24 h
* **
*
*
*
*
*
FIGURE 18.4 Pulmonary inflammation in particulate-exposed rats and controls as evidenced by % neutro-phils (PMN) in BAL fluids at 24 h, 1 week, 1 month, and 3 months postexposure (pe) Values given are meansGS.D Intratracheal instillation exposures of several particle-types produced a short-term, pulmonary inflammatory response, as evidenced by an increase in the percentages/numbers of BAL-recovered neutro-phils, measured at 24 h postexposure However, only the exposures to quartz particles (1 and 5 mg/kg) produced sustained pulmonary inflammatory responses, as measured through 3 months postexposure
*p!0.05
Trang 8Control 0.00 50.00 100.00 150.00 200.00 250.00
u/L
LDH
MTP
Alkaline phosphatase
300.00 350.00 400.00 450.00
1 mg/kg
5 mg/kg
1 mg/kg
5 mg/kg
1 mg/kg
5 mg/kg
1 mg/kg
5 mg/kg
Carbonyl iron
3 month
1 month
1 week
24 h
*
Control 0.00 10.00 20.00 30.00 40.00 50.00
mg/dL
60.00
1 mg/kg
5 mg/kg
1 mg/kg
5 mg/kg
1 mg/kg
5 mg/kg
1 mg/kg
5 mg/kg
Carbonyl iron
3 month
1 month
1 week
24 h
*
*
*
*
*
Control 0.00 20.00 40.00 60.00 80.00 100.00
u/L 120.00 140.00 160.00 180.00 200
1 mg/kg
5 mg/kg
1 mg/kg
5 mg/kg
1 mg/kg
5 mg/kg
1 mg/kg
5 mg/kg
Carbonyl iron
3 month
1 month
1 week
24 h
A
B
C
FIGURE 18.5 (See facing page.)
Trang 9Similarly, transient increases in BAL fluid microprotein (MTP) values were measured in the lung
different from control values at 1 week postexposure In contrast, exposures to 5 mg/kg a-quartz particles produced persistent increases in BAL fluid microprotein values at 24 h, 1 week, 1 month,
substantial increases in BAL fluid ALP values were measured at 1 week through 3 months post-exposure in rats exposed to 5 mg/kg a-quartz particles (Figure 18.5C)
To summarize the results from BAL fluid biomarker studies, intratracheal instillation exposures
to a-quartz particles resulted in sustained, dose-dependent, lung inflammatory responses, associ-ated with cytotoxic and lung permeability effects, measured from 24 h through 3 months
transient pulmonary inflammatory responses, and these effects were not sustained Exposures to
24 h postexposure; however, this was in large part related to the bolus dose associated with the intratracheal instillation exposure methodology
18.3.3.3 Pulmonary Histopathological Evaluations
Histopathological analyses of lung tissues revealed that pulmonary exposures to carbonyl iron, to
adverse effects when compared to PBS-exposed controls, as evidenced by the normal lung architecture observed in the exposed animals at postinstillation exposure time periods ranging
tissue section of a rat instilled with 5 mg/kg carbonyl iron particles at 1 month postexposure demonstrated appropriate alveolar macrophage phagocytic responses and normal lung
particle-exposed rats at each postexposure time period, and demonstrated normal pulmonary architecture
Histopathological analyses of lung tissues in rats exposed to a-quartz particulates in rats revealed that pulmonary exposures produced dose-dependent pulmonary inflammatory responses characterized by neutrophils and accumulations of foamy (lipid-containing) alveolar macrophage
In addition, lung tissue thickening as a prerequisite to the development of fibrosis was observed and progressive over postexposure time periods (Figure 18.6C and Figure 18.6D)
18.4 DISCUSSION
This chapter presents a case study and methodology designed to investigate the hazard potential of
FIGURE 18.5 BAL fluid analyses for particulate-exposed rats and corresponding controls at 24 h, 1 week,
1 month, and 3 months postexposure (pe) (A) LDH (lactate dehydrogenase), (B) MTP (microprotein values), and (C) alkaline phosphatase Values given are meansGS.D Transient and reversible increases in BAL fluid
postexpo-sure In contrast, exposures to 5 mg/kg a-quartz particles produced sustained increases in BAL fluid LDH values through the 3-month postexposure period *p!0.05
Trang 10compared with other negative control, reference particle-types such as R-100 fine-TiO2particles or carbonyl iron particles The combination of BAL and lung tissue studies concomitant with an experimental design consisting of dose-response, time-course evaluations, and the inclusion of reference particle-types provides a powerful tool for assessing the acute pulmonary toxicity of this new particle-type
The bioassay described herein provides evidence that toxicological information on a particle’s various surface treatments can be assessed in a routine systematic manner A beneficial feature of the bioassay is the ability to compare, via bridging strategies, the effects of inhaled versus instilled particulate materials In this regard, pulmonary bridging studies can generate important preliminary hazard data when assessing the safety of new developmental or commercial compounds or when making modifications to existing chemical products, such as surface coatings on particulates The strength of the bridging strategy is dependent upon having good inhalation toxicity data for comparisons to the instillation data The materials for which there is inhalation data can then be used as control particle-types for comparing with an intratracheal instillation bridging study (Figure 18.1) The basic idea for the bridging concept is that the effects of the instilled material serve as a control (known) material and then are “bridged” to the inhalation toxicity data for that material, as well as to the new materials being tested The results of bridging studies in rats are then useful as preliminary pulmonary toxicity screening (i.e., hazard) data, because consistency in the response of the inhaled and instilled control material serves to validate the responses with the newly
TB
AD
AD
TB
AD AD
A
C
B
D
FIGURE 18.6 Light micrographs of lung tissue from a rat exposed to (A) carbonyl iron particles (5 mg/kg) at
(A) and (B), these micrographs illustrate the terminal bronchial (TB) and corresponding alveolar ducts (AD) and demonstrate the lung architecture and normal macrophage phagocytosis of each particle (arrows) For (C) and (D), the arrows demonstrate accumulation of foamy alveolar macrophages in the alveolar regions of quartz-exposed rats