TOXICOLOGICAL SCIENCES 94(1), 193–205 (2006)
doi:10.1093/toxsci/kfl087
Advance Access publication August 23, 2006
The Spontaneously Hypertensive Rat: An Experimental Model of
Sulfur Dioxide–Induced Airways Disease
Urmila P. Kodavanti,*,1 Mette C. Schladweiler,* Allen D. Ledbetter,* Roselia Villalobos Ortuno,† Marie Suffia,†
Paul Evansky,* Judy H. Richards,* Richard H. Jaskot,* Ronald Thomas,* Edward Karoly,‡ Yuh-Chin T. Huang,‡
Daniel L. Costa,* Peter S. Gilmour,§ and Kent E. Pinkerton†
*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; †Center for Health and the Environment,
University of California, Davis, California, 95616; ‡Human Studies Division, National Health and Environmental Effects Research Laboratory, Office of
Research and Development, U.S. Environmental Protection Agency, Chapel Hill, North Carolina, 27599; and §Center for Environmental Medicine,
Asthma and Lung Biology, School of Medicine, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, 27599
Received June 12, 2006; accepted August 17, 2006
Chronic obstructive pulmonary disease (COPD) is characterized
by airway obstruction, inflammation, and mucus hypersecretion,
features that are common in bronchitis, emphysema, and often
asthma. However, current rodent models do not reflect this human
disease. Because genetically predisposed spontaneously hypertensive (SH) rats display phenotypes such as systemic inflammation,
hypercoagulation, oxidative stress, and suppressed immune function that are also apparent in COPD patients, we hypothesized that
SH rat may offer a better model of experimental bronchitis. We,
therefore, exposed SH and commonly used Sprague Dawley (SD)
rats (male, 13- to 15-weeks old) to 0, 250, or 350 ppm sulfur dioxide
(SO2), 5 h/day for 4 consecutive days to induce airway injury. SO2
caused dose-dependent changes in breathing parameters in both
strains with SH rats being slightly more affected than SD rats.
Increases in bronchoalveolar lavage fluid (BALF) total cells and
neutrophilic inflammation were dose dependent and significantly
greater in SH than in SD rats. The recovery was incomplete at
4 days following SO2 exposure in SH rats. Pulmonary protein
leakage was modest in either strain, but lactate dehydrogenase
and N-acetyl glucosaminidase activity were increased in BALF
of SH rats. Airway pathology and morphometric evaluation of
mucin demonstrated significantly greater impact of SO2 in SH
than in SD rats. Baseline differences in lung gene expression
pattern suggested marked immune dysregulation, oxidative stress,
impairment of cell signaling, and fatty acid metabolism in SH rats.
SO2 effects on these genes were more pronounced in SH than in
Disclaimer: The research described in this article has been reviewed by the
National Health and Environmental Effects Research Laboratory and U.S.
Environmental Protection Agency and approved for publication. Neither does
approval signify that the contents necessarily reflect the views and the policies
of the Agency nor does mention of trade names or commercial products
constitute endorsement or recommendation for use.
1
To whom correspondence should be addressed at Pulmonary Toxicology
Branch, MD: B143-01, Experimental Toxicology Division, National Health
and Environmental Effects Research Laboratory, U.S. Environmental Protection Agency, 109 TW Alexander Drive, Research Triangle Park, NC 27711.
Fax: 919-541-0026. E-mail: [email protected].
Published by Oxford University Press 2006.
SD rats. Thus, SO2 exposure in SH rats may yield a relevant
experimental model of bronchitis.
Key Words: chronic obstructive pulmonary disease; bronchitis;
spontaneously hypertensive rats; sulfur dioxide exposure; mucus
hypersecretion; inflammation.
Chronic obstructive pulmonary disease (COPD) results primarily from cigarette smoking and is the fourth leading cause
of mortality worldwide (Spurzem and Rennard, 2005). Although nearly 90% of COPD patients are smokers, only 10%
of the smokers develop the disease, suggesting that genetic
predisposition is a dominant factor (Siafakas and Tzortzaki,
2002). A variety of candidate genes have been associated with
disease susceptibility. Gene variants of a-1 antitrypsin expression show high risk for damage to alveolar structure. However,
bronchitis and COPD generally appear to have multigene
involvement (Barnes, 1999; Sandford et al., 2002). Smoker’s
lung disease is characterized by chronic active inflammation,
airway mucus hypersecretion, and emphysema (MacNee,
2005). It is only partially reversible upon cessation of smoking
(Rennard, 2005). Animal models exposed to cigarette smoke
and other chemicals have been studied to investigate disease
pathogenesis and therapeutic interventions, however, with only
limited success (Foronjy et al., 2006; Kodavanti et al., 1998,
Kodavanti and Costa, 2001; Shapiro, 2000; Vlahos et al., 2006).
Commonly used rat and mouse strains demonstrate only mild
inflammation and mucus secretion in response to cigarette
smoke (Guerassimov et al., 2004) or sulfur dioxide (SO2)
exposures (Kodavanti et al., 2000a; Lamb and Reid, 1968),
and the corresponding injuries are rapidly reversible. It has
been suggested that healthy laboratory rodents possess a remarkable ability to compensate and regenerate lung function
and structure following an injury (Costa et al., 1983), which
may underlie their relative insensitivity to experimental COPD.
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These shortcomings have slowed the progress in therapeutic
interventions, while the search continues for better experimental models of COPD.
Chronic bronchitis generally is a major attribute of COPD
and is characterized by airway inflammation and mucus hypersecretion (Rennard, 2005). Experimental models of bronchitis have been pursued using long-term SO2 exposures in
rats and dogs (Killingsworth et al., 1996; Knauss et al., 1976;
Lamb and Reid, 1968; Long et al., 1997, 1999; Shore et al.,
1987, 1995). SO2 is an upper respiratory tract irritant and
upon inhalation at high concentrations is known to induce
neutrophilic inflammation and airway mucus hypersecretion.
The mechanism of SO2-induced mucus secretion and inflammation seems to involve neurogenic substances such as
NK-1 and substance P (Killingsworth et al., 1996; Long et al.,
1997). Involvement of oxidative stress also has been suggested
(Gumuslu et al., 2001; Meng, 2003). Airways inflammation
have been associated with chemokine expressions in mice
(Meng et al., 2005) and increased airway hyperresponsiveness
in Sprague Dawley (SD) rats (Shore et al., 1995). All these
impairments are also apparent in human bronchitic patients
(Grootendorst and Rabe, 2004; Spurzem and Rennard, 2005).
Thus, these effects caused by SO2 in SD rats have been correlated with chronic bronchitis in humans, but the severity
and persistence of these effects are not similar to those seen
in human disease. It is likely that the mechanism by which
SO2 induces bronchitis is different from causation of bronchitis
in humans and that only limited injury is likely in healthy rat
strains.
We hypothesized that as only a predisposed subgroup of
humans acquire environmental disease, laboratory rodents
likewise require unique genetic susceptibility to develop analogous experimental disease. Because the spontaneously hypertensive (SH) rats exhibit genetic risk of cardiac disease and
phenotypic features of lung predisposition to injury (Kodavanti
et al., 2000c), we hypothesized that these rats may be uniquely
susceptible to develop a model of COPD with SO2. Although
every genetic component that plays a role in the development
of systemic hypertension has not been identified, SH rats
are known to demonstrate phenotypes such as systemic oxidative stress (Kobayashi et al., 2005), inflammation (SchmidSchonbein et al., 1991), hypercoagulation (Amagasa et al.,
2005), immunosuppression (Khraibi et al., 1984), and borderline pulmonary hypertension (Aharinejad et al., 1996), all
attributes also common in COPD (Sandford et al., 2002). The
purpose of this study was to characterize and compare SO2induced pulmonary and airway disease in SD and SH rats
using physiological, pathological, and molecular measures. We
compared SO2-induced pulmonary injury and inflammation,
airway mucus production, airway and lung pathology, and lung
tissue gene expression in SH as well as in commonly used
SD rats. We report that SH rats demonstrate remarkable
susceptibility to SO2-induced pulmonary inflammation and
mucus hypersecretion that persists much longer when com-
pared to SD rats. Differences in gene expression patterns in
both of these strains provide mechanistic insight for greater
susceptibility of SH than SD rats.
METHODS AND MATERIALS
Animals
Healthy, male, 12- to 15-weeks old, SD and SH (SHR/NCrlBR) rats were
purchased from Charles River Laboratories, Raleigh, NC. All rats were
maintained in an isolated animal room in an Association for Assessment and
Accreditation of Laboratory Animal Care approved animal facility (21 ± 1C,
50 ± 5% relative humidity, and 12-h light/dark cycle) for 1- to 2-week quarantine
and nonexposure periods. The rats were housed in plastic cages with beta-chip
bedding. All animals received standard (5001) Purina rat chow (Brentwood,
MO) and water ad libitum except during the daily exposure periods of 5 h. The
Environmental Protection Agency’s (EPA’s) Animal Care and Use Committee
approved the protocol for the use of rats in these inhalation studies.
SO2 Exposures
Each rat strain was randomized by body weight into three groups and then
further divided into two time points. In order to avoid acute ocular injury from
SO2 in the whole-body exposures, we conducted exposures by nose-only
inhalation. Concentrations of 200–500 ppm have been used previously in
inducing experimental bronchitis in rodents and other laboratory animals
(Knauss et al., 1976; Kodavanti et al., 2000b; Long et al., 1999; Shore et al.,
1987). Rats were acclimatized once to nose-only tubes prior to their air or SO2
exposures. Rats were restrained in nose-only tubes, and then tubes were
arranged in racks placed in stainless steel Hazelton 2000 inhalation exposure
chambers. Rats were then exposed to either filtered air or SO2 at 250 or
350 ppm, 5 h/day for 4 consecutive days. Anhydrous SO2 (99.8% purity,
National Welders, Charlotte, NC) was delivered into the inlet of the chamber
using a mass flow controller (Tylan FC-280, Mycrolis, Billerica, MA). The SO2
concentration in each chamber was monitored by an infrared gas analyzer
(MIran 1a, ThermoElectron, Waltham, MA) (247 ± 15 ppm and 353 ± 11 ppm).
Chamber relative humidity was 47–50%, temperature 23C, and chamber
airflow was ~ 550 l/min.
Whole-Body Plethysmograph Data Acquisition and Analysis
Barometric plethysmography using a whole-body plethysmograph (WBP)
system (Buxco Electronics, Inc, Sharon, CT) was employed to obtain data on
breathing parameters prior to and after exposure. The use of this methodology
permitted continuous monitoring of a number of ventilatory parameters, including breathing frequency (f), tidal volume (TV), minute ventilation (MV),
peak expiratory flow (PEF), peak inspiratory flow (PIF), inspiratory time (TI),
expiratory time (TE), pause (Pau), and enhanced pause (Penh). It should be
noted that TV is not directly measured but represents a value calculated using
the WBP system software. Since the system is calibrated over the normal physiological range, these values have been shown to accurately reflect direct
measurements of this parameter (Tankersley et al., 1997).
Each of the four WBP chambers (Model PLY3213, Buxco Electronics, Inc.,
Sharon, CT) was calibrated prior to analysis. A bias flow regulator drew
approximately 2 l/min of room air through each of the cylindrical chambers to
prevent carbon dioxide buildup within the WBP chambers. Unrestrained, freely
moving animals were placed in individual WBP and allowed for 1 min to settle
down followed immediately by 5 min of data collection. Each animal was
assessed several times prior to the start of first exposure and then at each time
prior to the start and at the end of SO2 exposure. Assessment was also done at 1
and 4 days postexposure. Diurnal variation was apparent in the breathing
parameters, and the values are given for only morning measurements. To
prevent diurnal variability, preexposure analyses were conducted during the
same time of day as the anticipated postexposure analyses. An analysis protocol
SULFUR DIOXIDE–INDUCED AIRWAYS DISEASE
was designed to ensure that animals from each group were equally distributed
among the WBP chambers. Data were collected for each parameter every 10 s
and averaged over 1-min intervals using vendor-supplied software (BioSystem
XA, Buxco Electronics, Inc., Sharon, CT). Automated breath-by-breath analyses
were performed using a rejection algorithm to eliminate breaths that were
outside a given range. Animals were taken out of the chambers after completion
of the 6-min protocol.
Necropsy, Sample Collection, and Analysis
One or four days following the last exposure, rats were weighed and
anesthetized with an overdose of sodium pentobarbital (50–100 mg/kg, ip).
From the first group of rats, the trachea was cannulated and the left lung
bronchus was tied. The right lung was lavaged with Caþþ/Mgþþ-free
phosphate-buffered saline (pH 7.4) with a volume equal to 35 ml/kg body
weight (near total lung capacity) 3 0.6 (right lung representing 60% of total
lung mass). Three quick in-and-out washes were performed with the same
buffer aliquot to enrich for protein and enzymes. This bronchoalveolar lavage
fluid (BALF) was collected in tubes and kept on ice for further analysis.
Lavaged lung lobes were quick-frozen in liquid nitrogen for RNA extraction.
The left lung was inflated with 10% neutral formalin, at 20 cm water pressure
for 30 min, and then tied and immersed in formalin for histology.
BALF Analysis for Determining Lung Injury
One aliquot of whole lavage fluid was used for determining total cell
counts (Coulter Counter, Coulter, Inc, Miami, FL), and a second aliquot was
centrifuged (Shandon 3 Cytospin, Shandon, Pittsburgh, PA) to prepare cell
differential slides. The slides were dried at room temperature and stained with
LeukoStat (Fisher Scientific Co, Pittsburgh, PA). Macrophages and neutrophils
were counted using light microscopy (over 200 cells counted per slide). The
remaining BALF was centrifuged at 15003 g to remove cells, and the supernatant fluid was analyzed for markers of lung injury and inflammation.
Cell-free BALF was analyzed for protein (lg/ml) content using Coomassie Plus
Protein Assay Kit (Pierce, Rockford, IL) and BSA standards from SigmaAldrich Chemicals (St Louis, MO). BALF samples were analyzed for albumin
content (lg/ml) using a commercially available kit (Diasorin, Stillwater, MN).
Lactate dehydrogenase (LDH) activity (U/l) was determined using a Kit (#228)
and standards from Sigma Chemical Co. N-Acetyl glucosaminidase (NAG)
activity (U/l) was measured using a commercially available kit and standards
from Roche Diagnostics (Indianapolis, IN). These assays were modified and
adapted for use on the Hoffmann-La Roche Cobas Fara II clinical analyzer.
Histopathology and Mucin Quantification
Lung tissues were processed, embedded in paraffin, sectioned at 5 lm, and
stained with hematoxylin and eosin (H&E) for pathological evaluation and
semiquantitative scoring of lesions. Severity scores (1 ¼ minimal, 2 ¼ slight/
mild, 3 ¼ moderate, 4 ¼ moderately severe, and 5 ¼ severe) were assigned
for a variety of pathological indices. Mean severity was determined by adding
severity scores of all animals within a group and then dividing the sum by the
total number of animals.
Additional serial sections from each lung were stained separately with alcian
blue and periodic acid–Schiff (AB-PAS) to distinguish mucus-secreting cells
on a background of H&E-stained epithelial cells. Specifically, the percentage
of airway epithelium with mucin was determined. Measurements were done
by estimating the percentage of the epithelial layer that stained positive for
AB-PAS (mucin) to the nearest 5%. This initial qualitative assessment permitted evaluation of patterns for different SO2 exposure levels. For quantitative
analysis of epithelial mucosubstances, 10 airway fields at 325 magnification
of the main axial pathway between three and six generations of airway branching were examined for each animal. Images taken using AB-PAS–stained slides
implemented a software program (Image J, version 1.240) and an image capture
software system. Digital images in color were taken at an image setting of
32 bit to quantify the pixel volume of mucosubstances within the airway
epithelial lining of each field. Also measured in the Image J and National
Institute of Health Image was the basal lamina length within each field to
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express pixel area (mucosubstance) to linear length (basal lamina). These
two measures were subsequently converted to mucosubstance volume/basal
lamina surface area for each airway observed. Statistical analyses of these results were done using the computer program StatView 5.0. The ANOVA test
with a p value of less than 0.05 (p < 0.05) was used to determine statistical
significance of the results.
RNA Isolation
Total RNA was isolated from the apical lung lobes snap-frozen in liquid
nitrogen. The frozen tissue was homogenized in TriReagent (Sigma), processed, and dissolved in 50 ll diethylpyrocarbonate-treated water. The quality
and quantity of RNA were assessed by using an Agilant Bioanalyzer (Agilent
2100 Bioanalyzer, Agilent Technologies, Palo Alto, CA).
Microarray Analysis
Microarray data were collected at Expression Analysis, Inc. (http://www.
expressionanalysis.com; Durham, NC) using the GeneChip Rat 230A Array
(Affymetrix Inc., Santa Clara, CA) containing 15,923 probe sets with known
genes including expressed sequence tags. After cRNA production, the quality
and quantity of each sample were assessed using a 2100 BioAnalyzer (Agilent
Technologies). The target cRNA was hybridized according to the ‘‘Affymetrix
Technical Manual.’’ A brief description of the process for cRNA synthesis,
hybridization, visualization, and quantification are described elsewhere
(Gilmour et al., 2006).
Real-Time Quantitative PCR
RNA was DNase treated and one-step RT-PCR was carried out using
‘‘Platinum Quantitative RT-PCR ThermoScript One-Step System’’ (Invitrogen,
Carlsbad, CA) following the protocol recommended for the kit. All reactions
were run using 100 ng of total RNA. Reactions were multiplexed, that is,
containing one pair of target gene primers and one pair of endogenous control
gene (b-actin) primers in each sample well. Real-time PCR was conducted on
an ABI Prism 7900 HT Sequence Detection System (Applied Biosystems, Foster
City, CA). RT-PCR conditions consisted of reverse transcription for 20 min
at 58C and inactivation of reverse transcriptase activity for 5 min at 95C.
There were 40 amplification cycles at 95C for 15 s followed by 60C for 60 s.
Primers were Lux (Invitrogen, Carlsbad, CA) labeled by FAM (target genes) or
by JOE (control gene, b-actin) and were purchased from Invitrogen. The
sequences were (1) macrophage inflammatory protein-2 (MIP-2) 5#-CGGGCAGAAATAACAGTCGTCC-3# and 5#-CGGGCAGAAATAACAGTCGTCC3#, (2) TNF-a 5#-CGAGTGGGCTACGGGCTTGTCACT-3# and 5#-GGGCTCCCTCTCATCAGTTCC-3#, (3) heme oxygenase-1 (HO-1) 5#-CGTAAAGCGTCTCCACGAGGT-3# and 5#-CGCTAGAGAGGTCACCCAGGTAG-3#, and
(4) b-actin 5#-CACACTTCGCAGCTCATTGTAGAAAGTG-3# and 5#-TTGAACACCGGCATTGTAACCAA-3#. Data were analyzed using ABI Sequence
Detection System software version 2.1. For each plate run, the cycle threshold
(cT) was set to about one order of magnitude above background fluorescence,
and cT values were obtained for each sample. In each case, cT of the target
gene was normalized to changes in actin cT to account for variability in starting
RNA amount. Expression for each exposure condition was quantified relative
to expression of the corresponding saline control group.
Statistical Analysis
The data for breathing parameters were analyzed using repeated measure mix model ANOVA of SAS software program (SAS, Cary, NC). The
BALF data were analyzed using a multivariate ANOVA model (SAS). The
independent variables were strain (at two levels: SD and SH), exposure (at
three levels: 0, 250, and 350), and day (at two levels: 1 and 4). In cases where
the ANOVA assumptions were not satisfied, a transformation of the data was
performed in order to meet these requirements. If the transformations failed
to satisfy the assumptions, a distribution-free method was used for the analysis.
Subsequent to the overall analysis, pairwise comparisons were performed
as subtests of this analysis. No adjustment was made to the level of significance
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due to multiple comparisons. PCR data (delta-cT values were considered as
opposed to fold change over control) were analyzed by a two-way ANOVA
with SO2 exposure as one factor and rat strain as the other using SigmaStat
software version 3.1 (SPSS, Inc, Chicago, IL). In the case of significant
interaction, step-down ANOVAs were used to test for main effects with
time. Pairwise comparisons between groups were made using Fisher’s least
significant difference test. The accepted level of significance for all tests
was p < 0.05.
Analysis for gene chip data. Affymetrix CEL data files were imported
into R, an open source statistical scripting language (http://www.R-project.org)
used in conjunction with the Bioconductor project (http://www.bioconductor.
org) (Gentleman et al., 2004). Normalized values with RMA background correction, quantile normalization, and median polish were calculated with the
R/bioconductor package AffylmGUI (Irizarry et al., 2003).
RMA normalized values were imported into the MultiExperimentViewer
(TMEV4, The Institute of Genomic Research, http://www.TIGR.org) for statistical analysis (Irizarry et al., 2003; Saeed et al., 2003). Two-way ANOVA was
used to identify differentially expressed genes by strain, exposure, or strainexposure interaction at a p value of < 0.01. Resulting gene lists were then
filtered to remove nonannotatable probe sets.
A heat map was generated with the mean values of the four biological
replicates for each strain and exposure normalized to the mean value of the SD
air group. A hierarchical clustering analysis based upon average linkage and
Euclidian distances was then used to cluster genes on the y-axis and strain/
exposures on the x-axis using TMEV4.
The microarray data were deposited in the Gene Expression Omnibus Web
site (http://www.ncbi.nlm.nih.gov/geo/) (accession number GSE4702).
RESULTS
Body Weight Changes and Breathing Parameters
Restraining rats in nose-only inhalation tubes for 5 h during
acclimatization followed by a 4-day air exposure period caused
nearly 5% weight loss in SH but not in SD rats. SO2 exposure
was associated with further weight loss in a concentrationdependent manner in both strains (Fig. 1). Unlike previously
observed baseline differences in f, MV, TV, PIF, and PEF
between Wistar Kyoto (WKY) and SH rats (Kodavanti et al.,
2005), there were no significant differences in these breathing
FIG. 1. Comparative body weight gain (g) in SD and SH rats following 4day exposure to SO2. Rats were exposed to filtered air or SO2 5 h/day 3 4 days.
Values represent total weight gain (g) from first morning before their start of
exposure to necropsy (means of 13–14 rats per group). ‘‘*’’ Indicates significant
difference from time matched control, and ‘‘#’’ indicates significant strain
difference at matching concentration.
parameters between air control SD and SH rats prior to
restraining in tubes (Fig. 2). However, air-exposed control SD
rats while restrained in the tubes did exhibit altered breathing
parameters (a decrease in f, MV, PIF, and PEF). Exposure to SO2
in SD rats caused further decreases in f and MV. There were also
increases in TV, TI, TE, RT, and PenH following SO2 exposure
in SD rats (Fig. 2). Because the air-exposed SH rats did not
demonstrate significant changes in breathing parameters as
a result of nose-only tube restraint, the changes induced by SO2
exposure in these rats were more clearly distinguishable between
control and SO2 exposure. SH rats demonstrated marked and
consistent decreases in f, MV, and PIF while increases occured
in TV, TI, TE, RT, Pau, and PenH (Fig. 2). Some of these
differences were found not to be significant when individual
groups were considered using adjusted p values; however, they
did demonstrate significant difference when considered collectively or with unadjusted p values. It is noteworthy that the
changes in breathing parameters occurred in the same direction
in both rat strains, although there were quantitative differences.
The overall effect of SO2 plus tube restraint in SD rats was
comparable to the effect of SO2 alone in SH rats.
BALF Analysis of Lung Inflammation and Injury
These parameters were determined by analysis of BALF at
1 or 4 days following 4-day exposure to air or SO2. Total
lavageable cells increased significantly 1 day postexposure
only in SH rats, while no increase occurred in SD rats (Fig. 3).
The increase in cells persisted until 4 days postexposure.
The increase in total cells was accounted partly by a small
but concentration-dependent increase in alveolar macrophages
(AMs) in SH rats at 1 day postexposure. However, AM returned
to control 4 days postexposure. No changes occurred in SD
rats. The increase in total cell number following SO2 exposure
was due to the increased (~403) neutrophils in SH rats
(Fig. 3). This increase persisted up to 4 day postexposure in
rats exposed to 350 ppm SO2. Unlike the increase in SH
rats, SD rats demonstrated only a threefold increase in the
neutrophils at the highest SO2 concentration at 1 day but not at
4 day postexposure.
Since SO2 effects are likely to occur primarily in the airways, the total lavageable protein and albumin, markers of
alveolar protein leakage in the lung, did not increase significantly in SD or SH rats following 1 or 4 days after exposure
(Fig. 4). However, LDH activity and NAG activity was moderately increased in BALF of SH rats suggesting cytotoxicity.
This increase may have resulted from cytotoxicity in the airways with activation of resident macrophages as opposed to
effects on deep lung parenchyma. SD rats did not demonstrate
any SO2-induced increases in LDH or NAG activity.
Histological Evaluation and Mucus Quantification
A detailed histopathological analysis was done on respiratory tract tissues to determine the type and patterns of
SULFUR DIOXIDE–INDUCED AIRWAYS DISEASE
197
FIG. 2. Comparative evaluation of breathing parameters in SD and SH rats prior to and during exposure to SO2. All measurements were performed in
the morning of each day using Buxco whole-body plethysmography. The measurements of day 1 (D1) show values prior to the start of SO2 exposure. Rats
were exposed to filtered air or SO2 5 h/day 3 4 days. Values represent means of 17 SD or 17 SH rats for day 1 (D1) and five to eight rats per group thereafter.
Note that Pause and PenH are unitless values. Standard errors are not placed on bars for improving clarity of data. The adjusted < 0.05 p values were as follows
for comparisons: f: 0 ppm different from 250 and 350 ppm for SH rats at D3, D4, and D4 þ 1; MV: 0 ppm different from 350 ppm for SH rats at D3, D4, and
D4 þ 1; TI: 0 ppm different from 350 ppm for SD and SH rats at D3 and D4 and for only SH at D4 þ 1; PIF: 0 ppm different from 350 ppm for SH rats at D3, D4,
and D4 þ 1; and SD-0 ppm is different from SH-0 ppm at D4; all other comparisons were not significantly different when adjusted p values were considered.
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5E–5H. Epithelial cell hyperplasia and the presence of inflammatory cells were clearly evident in SO2-exposed SH
(Fig. 5F) and SD rats (Fig. 5H) relative to air-exposed SH
(Fig. 5E) and SD rats (Fig. 5G). The presence of inflammatory
cells within the airways of both the SD and SH rats was
indicative of airway effects (Fig. 5).
Quantitative histological scoring of pathological lesions of
the lung confirmed SO2-induced bronchiolar epithelial cells
hyperplasia, peribronchiolar inflammation, vascular mineralization, and increased goblet cells (Table 1). Morphological
quantitative analysis of mucin volume in airways indicated
marked and concentration-dependent increase in SH and
SD rats following SO2 exposure. The severity of increase was
more pronounced in SH rats (Fig. 6).
Gene Expression Analysis Using Real-Time PCR
Real-time PCR was performed for pulmonary mRNA expression of MIP-2, TNF-a, and HO-1 in order to correlate
inflammatory changes with expression of inflammatory cytokine genes. The expression of cytokine genes was less dramatically induced than the apparent neutrophilic inflammation
in the lung (Fig. 7). Only a 3-fold increase in MIP-2 and 2.5fold increase over control in TNF-a expression occurred in
SH rats with no changes in SD rats. HO-1 expression was not
significantly affected by SO2 exposure in either strain.
Gene Expression Analysis Using Microarray
FIG. 3. Inflammatory changes in the lungs of SD and SH rats following
4-day exposure to SO2. Rats were exposed to filtered air or SO2 5 h/day 3 4 days,
and BALF was analyzed for inflammation 1 or 4 days later. Total cells were
counted using coulter counter, and individual cell types were identified from
stained cytospin slides of BALF. Values represent means ± SE of five to eight
rats per group. ‘‘*’’ Indicates significant difference from time matched control,
and ‘‘#’’ indicates significant strain difference at matching concentration.
pathological changes and mucus secretion in the airways of SD
versus SH rats. Figure 5 demonstrates representative changes
in the airways of SO2-exposed SD and SH rats. Lung sections
stained with AB-PAS in air-exposed SH (Fig. 5A) or SD
(Fig. 5C) rats demonstrated minimal mucus cells in the airways. SO2-exposed SH rats demonstrated more dramatic increases in cell numbers with blue stain suggesting increases
in the mucus content of goblet cells as well as cell numbers
(Fig. 5B). The increase in goblet cells was also evident in SD
rats following SO2 exposure. However, the magnitude of the
increase was less than that of the SH rats (Fig. 5D). Serial
sections of lung tissues stained with H&E are shown in Figures
Lung RNA was analyzed to get further insight into the
global gene expression profile with the goal to highlight rat
strain differences in SO2 effects. Expression changes associated
with oxidative stress, acute phase response, immune modulation, cell signaling, protease/antiprotease balance, and inflammatory gene markers were analyzed. Among the oxidative stress
genes, glutathione S-transferase expression in SH rats was
higher than in SD rats, whereas glutathione peroxidase gene
expression was lower (Fig. 8). Marked strain-related differences
existed in the baseline expression of immune and inflammatory
response genes. Many of which were upregulated several fold,
while others were downregulated in SH rats relative to agematched SD rats (Fig. 8). There were also marked strain differences in expression of several genes involved in cell signaling
in which SH rats exhibited downregulation in most cases.
A variety of differentially expressed genes could be placed in
these categories: proteases/antiproteases, oxidases, cytochrome
P450s, blood coagulation markers, mitochondrial function, fatty
acid metabolism, and endothelial function.
A heat map of identified genes that showed significant
interaction for SO2 effects between SD and SH rats is given in
Figure 8. Genes demonstrating interaction between rat strain
and SO2 effect were those that are primarily involved in
inflammatory and immune modulation. Some of these genes
also involved oxidative stress responsive protein markers and
mitochondrial respiration. CD36 antigen, beta defensin,
SULFUR DIOXIDE–INDUCED AIRWAYS DISEASE
199
FIG. 4. Lung injury as determined by analysis of biochemical markers in BALF in SD and SH rats following 4-day exposure to SO2. Rats were exposed to
filtered air or SO2 5 h/day 3 4 days. BALF was analyzed for injury markers, such as total protein, albumin, LDH, and NAG, 1 or 4 days later. Values represent
means ± SE of five to eight rats per group. ‘‘*’’ Indicates significant difference from time matched control, and ‘‘#’’ indicates significant strain differences at
matching concentration.
hypoxia upregulated 1, and MIP-alpha receptor genes were
among those which were downregulated in SH but upregulated
in SD rats following SO2 exposure. However, myxovirus resistance 1, catechol-o-methyltransferase, cAMP responsive
element binding protein-like 2, carbonic anhydrase 3, IL-1beta, and glutathione S-transferase mu 5 genes were among
those which were upregulated in SH and downregulated in SD
rats following SO2 exposure.
DISCUSSION
Based on our earlier work with the SH rat, we hypothesized
that this strain would exhibit greater responsiveness to irritant
SO2 than other conventional laboratory strains and might reveal
a better model for bronchitis in the rat. Our hypothesis centered
on the fact that the SH rat demonstrates phenotypic risk factors
of COPD, such as systemic inflammation and oxidative stress,
hypercoagulation, suppressed immune function, and borderline
pulmonary hypertension. Our earlier studies with irritant particles have shown that the SH rat to be more susceptible to
lung injury and inflammation than healthy Wistar Kyoto
rats (Kodavanti et al., 2000c, 2002). In this study, we examined
SO2-induced airway inflammation, mucus hypersecretion,
pathology, and gene expression patterns in the SH relative to
SD rats, conventionally used to study bronchitis. Four consecutive days of 5-h SO2 exposures induced highly significant
neutrophilic inflammation response and mucus production in
the airways of the SH relative to that of the SD rats. The strainrelated differences in inflammation could not be explained by
SO2-induced changes in breathing parameters measured before
or after exposure. The inflammatory response primarily
occurred in the airways as no pulmonary protein leakage or
alveolar pathology was noted in either strain. Four days after
SO2 exposure, the inflammatory response persisted in SH rats
but had subsided in SD rats. The mucus response similarly
persisted. The slow recovery may reflect the extent of initial
injury and inflammation caused by SO2 in SH rats. Gene
expression patterns indicated significant strain-related differences in SO2 effect among key inflammatory/immune modulatory and oxidative stress response genes between strains
following exposure. Our data suggest that SH rats may be
a better strain than SD rats for development of an experimental
model of SO2-induced bronchitis.
Since changes of breathing parameters can impact the deposition of SO2 in the airways and may influence the sensitivity
of rats to develop toxicity, we wanted to determine if significant
strain differences in breathing parameters were responsible
for the difference in the strain sensitivity. It should be noted
that the breathing parameters were not measured during actual
exposure but were measured immediately after the exposure
and next day with the assumption that the measurements at
these times will reflect the changes that are likely during exposure. Restraining SD but not SH rats in nose-only inhalation
tubes decreased f, PIF, and PEF but not TV and as a result
decreased MV. However, because the values of breathing
parameters were similar in SO2-exposed SD and SH rats
despite differences in air-exposed controls, it is unlikely that
differences in SO2 deposition accounted for greater effectiveness of SO2 in SH than in SD rats. There was no clear
dose-response as both doses affected rats similarly in most
parameters. Unlike the differences we observed between SH
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KODAVANTI ET AL.
FIG. 5. Representative histological staining of the lung sections for mucin and inflammation in SD and SH rats exposed to filtered air or SO2. The extent of
airway mucin secretion and inflammation was evaluated in rats at 1 and 4 days following last SO2 exposure. Serial lung tissue sections were stained with AB-PAS
(mucin) and H&E, and representative airway changes are shown in the figure. (A–D) show AB-PAS–stained lung sections (A, SH-air; B, SH-SO2; C, SD-air; and
D, SD-SO2) and (E–H) show H&E-stained serial lung section (E, SH-air; F, SH-SO2; G, SD-air; and H, SD-SO2) from air or SO2-exposed rats at 4 days following
last exposure. Asterisks in (B) and (D) show purple mucus staining of airway epithelial cells. Arrows in (F) and (H) show inflammatory cells in airways.
and WKY rats in breathing parameters in an earlier study
(Kodavanti et al., 2005) and those observed by other investigators (Debreczeni et al., 1989), SH rats did not differ
significantly from SD rats in baseline values of breathing
parameters. However, the effect of SO2 was more severe in
SH than in SD rats. It is not clear at this time if the changes in
breathing parameters relate to biological sensitivity to mucus
cell proliferation.
Chronic active airway inflammation and mucus hypersecretion are two primary pathological features of human bronchitis
and COPD (MacNee, 2005). Therefore, we wanted to thoroughly examine these changes qualitatively and quantitatively in
both rat strains to demonstrate the severity and persistence of
these features. Neutrophilic inflammation due to SO2 exposure
has been noted in earlier studies using SD rats (Killingsworth
et al., 1996; Kodavanti et al., 2000b), which is consistent with
our present finding. However, the inflammatory response was
mild and rapidly reversible upon termination of SO2 exposure
with mucus production being more persistent than inflammation (Kodavanti et al., 2000a). We report here that SH rats
mounted a remarkably greater inflammatory response with
mucus production more persistent than that of SD rats. Although several laboratory species have been used in developing
an SO2-induced bronchitis models (Killingsworth et al., 1996;
Shore et al., 1987, 1995) and their limitations are well reported
(Kodavanti et al., 2000a,b), no studies have compared more
201
SULFUR DIOXIDE–INDUCED AIRWAYS DISEASE
TABLE 1
The Mean Severity Grades for the Histopathologic Lesions in SD and SH Rats Following Air or SO2 Exposure
SD rats
Pathology indices
Macrophages, nonpigmented
Macrophages, pigmented
Bronchiolar epithelial hyperplasia
Peribronchiolar acute inflammation
Vascular mineralization
Goblet cells
SO2, 0 ppm
1.3
0.4
0.8
0.0
0.5
1.2
±
±
±
±
±
±
0.2
0.2
2.0
0.0
0.3
0.1
SO2, 250 ppm
1.9
0.5
0.5
0.2
1.4
1.5
±
±
±
±
±
±
0.2
0.2
0.2
0.2
0.3
0.2
SH rats
SO2, 350 ppm
1.3
0.5
0.5
0.5
1.6
1.7
±
±
±
±
±
±
0.2
0.2
0.2
0.3
0.2
0.2
SO2, 0 ppm
1.0
0.8
0.0
0.0
0.9
1.3
±
±
±
±
±
±
0.0
0.1
0.0
0.0
0.3
0.2
SO2, 250 ppm
1.3
1.1
1.1
0.3
0.4
2.6
±
±
±
±
±
±
0.2
0.1
0.1
0.2
0.3
0.2
SO2, 350 ppm
1.3
1.0
1.3
1.0
1.3
3.0
±
±
±
±
±
±
0.2
0.0
0.3
0.3
0.3
0.3
Note. The values indicate mean ± SE (n ¼ 10 rats per group; five at 1 day and five at 4 day postexposure) of pathology score assigned to each rat following
5 h/day 3 4-day SO2 exposure based on 1 ¼ mild, 2 ¼ moderate, 3 ¼ marked, and 4 ¼ severe. Note that based on H&E-stained slides evaluation, lesion severities
at 1 and 4 day postexposure were not different, and therefore, the values for both time points were combined.
than one strain in parallel to demonstrate differences in disease
severity. The comparative evaluation of SD and SH rats indicate
that the use of SH rats may offer more intense and persistent
bronchitic symptoms reminiscent of the human disease. Further
characterization of this model upon low concentration of
SO2 exposure for a longer period of time is likely to increase
persistency of inflammation.
Since SO2 is highly reactive and is hydrated very rapidly, it
is converted to sulfite and bisulfite as soon as it comes in contact with airway lining fluid while triggering modifications
in protein disulfide bonds (Petering and Shih, 1975). The exact
mechanisms that trigger neutrophil migration and increased
mucus secretion are unknown. C-fiber deactivation has been
proposed to be involved as more severe SO2-induced inflammation was noted in capsaicin-pretreated rats (Long et al.,
1997, 1999). It is not clear which mechanism may be responsible for exacerbated SO2-induced inflammation in SH
rats. The evaluation of gene expression pattern may provide
insights into mechanisms of exacerbated SO2 response in
SH rats.
The present experimental design does not allow for identifying the genetic risk factors associated with greater susceptibility of SH rats; however, evaluating global gene expression
profiles of strains in parallel may provide distinctive biological
pathways or functions. The striking baseline differences in
immune modulatory and inflammatory gene expression may
reflect compromised immune function in SH rats (Khraibi
et al., 1984). Immune deregulation may also be evident by
deficiency in beta defensin and elevated expression of inflammatory cytokine mRNA expression. Gene expression analysis
demonstrated reduced expression of beta defensin in the SH
as opposed to SD rats in the present study. Genetic variants
of human beta defensin have been associated with COPD
(Matsushita et al., 2002). Also, increased expressions of cytokines, IL-1-beta, MIP-2 (a human analog of IL-8), and TNF-a,
and iron-binding protein, transferrin, in inflammatory cells of
FIG. 6. Quantitative analysis of mucin levels in the airways of SD and
SH rats following 4-day exposure to SO2. Percentage of airway epithelium
containing mucin was determined by estimating the percentage of the
epithelial layer that stained positive for AB-PAS (mucin) relative to the total
epithelial layer area at 1 or 4 days following last SO2 exposure. Each group
represents mean ± SE of five rats. ‘‘*’’ Indicates significant difference from
time matched control, and ‘‘#’’ indicates significant strain difference at
matching concentration.
FIG. 7. Differential effect of SO2 exposure in lung mRNA expression for
inflammatory cytokines in SD and SH rats. Rats were exposed to filtered air or
SO2 5 h/day 3 4 days, and total lung RNA was analyzed by real-time
quantitative PCR to determine mRNA expression for cytokines. Only control
and 350 ppm SO2-exposed SD and SH rats at 1 day postexposure were
evaluated. Values represent mean ± SE (four rats per group). ‘‘*’’ Indicates
significant difference from time matched control, and ‘‘#’’ indicates significant
strain difference.
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KODAVANTI ET AL.
FIG. 8. Heat map showing at strain-specific differential gene expression as determined by Affymetrix gene chips in rats exposed to air or SO2. Total RNA was
reverse transcribed, labeled, and hybridized with Affymetrix rat 230A gene chips. Only control and 350 ppm SO2-exposed SD and SH rats were evaluated. Red and
green color intensities indicate increases and decreases, respectively, in expression of genes. Expression values of SD air-exposed rats were given a value of 1, and
all other data were normalized relative to SD air group. Note that only differentially expressed genes which were significant at p < 0.05 between SD and SH rats
following SO2 exposure are listed (interaction).
chronic bronchitic patients (Chung, 2001; Stites et al., 1998)
are consistent with observed SO2-induced expression changes
in SH relative to SD rats. Exposure to SO2 has been shown to
induce inflammatory response in rats (Long et al., 1999), but
systematic analysis of the role of cytokines has not been
addressed in rat model of bronchitis. Thus, one can assume that
some of the molecular changes leading to inflammation and
mucus hypersecretion in SH rats after acute SO2 exposure are
common in susceptible humans who develop chronic bronchitis. However, it is likely that the pathogenesis of irreversible
bronchitis resulting from years of cigarette smoke exposure
may involve multiple component-specific cell injuries and
complex interplay of injury and compensatory mechanisms.
SH rats harbor a deletion variant in the CD36 fatty acid
transporter, an integral membrane glycoprotein expressed on
the surface of cells, causing defective fatty acid metabolism,
insulin resistance, and hypertension (Hajri et al., 2001;
Pravenec and Kurtz, 2002; Pravenec et al., 2004). The nearly
absent expression of the CD36 gene in SH but high expression in SD rats confirm this deficiency and validates the quality
of data generated by the gene chip experiments in our study.
This defect in CD36 has been shown to be associated with
metabolic syndrome characteristics of hypertensive rats and
is thought to contribute to cardiac hypertrophy (Hajri et al.,
2001). However, it has been reported that transgene expression of CD36 in SH rats while ameliorating metabolic disturbances does not affect hypertension (Pravenec et al., 2003),
again supporting the hypothesis that multigenetic defects are
likely involved. The CD36 gene also has been shown to have
a major role in immune function and is thought to be involved
in inflammation associated with COPD (Pons et al., 2005).
Although the complex functional roles of CD36 are still
unclear, it is likely that increased susceptibility of SH rats to
SO2-induced inflammation may be in part due to their defect in
CD36 and altered immune regulation and metabolic function.
Examples of genes that were differentially expressed between SH and SD rats include RT1 class IB, an MHC class 1
antigen involved in natural killer cell activity (Leong et al.,
1999), cysteine-rich 61, involved in regulation of cell signaling
(Lombet et al., 2003), and telomeric repeat binding factor 2
203
SULFUR DIOXIDE–INDUCED AIRWAYS DISEASE
critical in aging and cancer (Gu et al., 2005). It is not known
why these genes may be downregulated in SH rats, but these
might represent genetic markers involved in their greater
susceptibility to environmental exposure. Clearly more investigation is needed.
Because SH rats begin their progressive hypertension at
12 weeks of age, their underlying sensitivity might reflect this
change. The present study does not allow separation of the role
of hypertension from other phenotypic and genetic factors.
Drew et al. (1983) used DahlS rats (sensitive to salt-induced
hypertension) and DahlR rats (resistant to salt-induced hypertension) and showed that the DahlR demonstrated a slight
decrease in blood pressure following SO2 exposure (50 ppm),
while DhalS rats demonstrated an increase. More studies will
be required to investigate the contribution of hypertension. The
purpose of this study was to investigate if SH rats demonstrated
greater susceptibility than conventionally used SD rats and, if
so, to validate the model at the physiological, pathological, and
molecular levels. In SH rats, efforts of reducing hypertension
also reduced underlying oxidative stress and vice versa (Lazaro
et al., 2005; Rodriguez-Iturbe et al., 2003), suggesting that the
hypertension phenotype is mechanistically linked to the presence of other ailments and may modify susceptibility to SO2.
Thus, careful experimentation will be required to determine
the role of hypertension versus other risk factors in understanding their susceptibility to SO2 exposure.
Although biochemical, molecular, and morphological features of bronchitis and COPD are well categorized in patients
(MacNee, 2005; Spurzem and Rennard, 2005), the pathogenic
mechanisms by which reactive cigarette smoke components
initiate the complex process of chronic active inflammation,
airway remodeling, mucus hypersecretion, and alveolar destruction are less well understood. Matrix metalloproteinases,
inactivation of protease inhibitors, and collagen and elastin
degradation by-products have been shown to play a role in
chronic activation of CD8 T lymphocytes, AMs, and neutrophils (Cawston et al., 2001; Postma and Timens, 2006). It is
not understood why some individuals develop both alveolar
as well as airway pathologies from cigarette smoke exposure as
in COPD, whereas others develop primarily an airways disease
as in bronchitis. It is likely that genetic and molecular differences between individuals determine the primary pulmonary
target tissues affected by chronic cigarette smoking. Because
high-level SO2 exposures, such as one used in the present study,
primarily cause damage to airway epithelial cells and mucus
hypersecretion without significant alveolar destruction, the
mechanism by which SO2 cause bronchitis is likely to be
different from that of tobacco smoke in humans and animals.
Nevertheless, the SO2-induced bronchitis in SH rats may allow
one to understand the role of airways disease and genetic
susceptibility. Also, because SH rats demonstrate pathologies
of persistent neutrophilic inflammation and mucus hypersecretion, they may provide a better model for therapeutic interventions. Based on the sensitivity of SH rats to SO2, we predict
that experimental exposure of these rats to tobacco smoke may
also yield a highly relevant animal model of bronchitis and
COPD. Studies are in progress to characterize cigarette smokeinduced pulmonary disease in SH relative to healthy rats.
Bronchitis and COPD are chronic diseases that develop
over several years of cigarette smoking (MacNee, 2005). Experimental animals are typically exposed to SO2 for 6–8 weeks
to develop features of human bronchitis (Kodavanti et al.,
2000b; Lamb and Reid, 1968; Shore et al., 1995). In this study,
we exposed rats to SO2 for only 4 consecutive days because
we initially wanted to determine relative susceptibility of two
strains to acute exposures at concentrations that have been
used previously in experimental bronchitis. Secondly, due to
the sensitivity of SH rats to SO2 at concentrations used in
the present study, we presumed that a longer duration exposure
may induce severe compromise of pulmonary health. A followup study will involve long-term episodic exposures to SO2 at
lower concentrations, which is hypothesized to provide longer
lasting pulmonary inflammation and mucus hypersecretion
than observed in this study.
In conclusion, we provide evidence that SH rats, which are
genetically predisposed to hypertension and exhibit associated
systemic risk factors also found in human COPD patients, are
more susceptible to SO2-induced airway inflammation and
mucus hypersecretion than conventionally used SD rats. Thus,
the SH rat appears to be a more relevant experimental human
bronchitis model than seen previously with other rats. Further,
pulmonary gene expression profiling in SH and SD rats following air and SO2 exposure allowed identification of genetic
markers which might be responsible for greater SO2 sensitivity.
These genetic targets might also underlie human susceptibility
to chronic bronchitis.
ACKNOWLEDGMENTS
We thank Mr Donald Doerfler, Ms Najwa Coates, and Ms Judy Schmid
(U.S. EPA) for statistical analysis of the data. We also thank Drs Linda
Birnbaum, Steve Gavett, Gary Hatch, and Andrew Ghio of the U.S. EPA for
their critical review of the article.
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