DTIC ADA553605: Pathological Studies on the Protective Effect of a Macrolide Antibiotic, Roxithromycin, against Sulfur Mustard Inhalation Toxicity in a Rat Model

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by  Defense Technical Information Center

ToxicologicPathology,39:1056-1064,2011
Copyright#2011byTheAuthor(s)
ISSN:0192-6233print/1533-1601online
DOI:10.1177/0192623311422079
Pathological Studies on the Protective Effect of a Macrolide
Antibiotic, Roxithromycin, against Sulfur Mustard
Inhalation Toxicity in a Rat Model
XIUGONG GAO1,DANA R. ANDERSON2, AMMONW.BROWN1, HSIULINGLIN1,JACK AMNUAYSIRIKUL1,AILEEN L.CHUA1,
WESLEY W.HOLMES2,AND PRABHATI RAY1
1WalterReed Army InstituteofResearch, SilverSpring, Maryland,USA
2U.S. Army MedicalResearch InstituteofChemical Defense, Aberdeen ProvingGround,Maryland,USA
ABSTRACT
Macrolideantibioticshavebeenshowntoprotectairwayepithelialcellsandmacrophagesfromsulfurmustard(SM)–inducedcytotoxicity.Inthe
currentstudy,theefficacyofroxithromycininamelioratingSM-inducedrespiratoryinjurywasfurtherevaluatedinaratmodel.Anesthetizedrats(N
¼8/group)wereintratracheallyexposedtoSMbyvaporinhalation.Forthedrugtreatmentgroups,ratswereorallygiven10,20,or40mg/kgrox-
ithromycinonehrpriortoexposureandeverytwenty-fourhrthereafter.Afterone,three,orsevendaysoftreatment,sectionsofthelungwereexam-
inedandscoredforhistopathologicalparameters.TreatmentwithroxithromycinamelioratedmanyofthesymptomscausedbySMinsomeanimals.
Inparticular,treatmentat40mg/kgforthreedaysshowedsignificantimprovements(p<.05)overtheuntreatedgroup.Whentheevaluationwas
focusedontrachea,treatmentwithroxithromycinforthreedaysshowedatrendofdose-dependentprotection;moreover,thegroupstreatedwith20or
40mg/kgofroxithromycinwerestatisticallydifferent(p<.001andp<.05,respectively)fromtheuntreatedgroup.Theseresultssuggestthatrox-
ithromycinprotectsagainstsomedamagesassociatedwithSMinjuryinthelung,particularlyintheupperrespiratorytract.
Keywords: sulfurmustard;respiratoryinjury;pathology;macrolideantibiotic;roxithromycin;ratlung;toxicity.
INTRODUCTION lung, including the alveoli. SM-induced epithelial cell injury
and cell death in the tracheobronchial tree leads to inflamma-
Sulfurmustard(2,2’-dichlorodiethylsulfide,SM)isachem-
tionandsloughingofthemucosaduringacutestages.Theclin-
icalwarfareagentthathasbeenusednumeroustimesinhistory
icalsignsofSMinhalationincludeasthma,chronicbronchitis,
sinceWorldWarI,causinginjuriesanddeathstoalargenum-
bronchiectasis, and pulmonary fibrosis (Emad and Rezaian
ber of victims (Szinicz 2005; Kehe and Szinicz 2005). Being
1997).Pulmonarydamageandassociatedsecondaryinfections
the major chemical warfare agent during World War II, SM
have been responsible for most fatalities (Papirmeister et al.
was producedandstockpiled bymany countriesand isproba-
1991).
bly still the most distributed chemical warfare agent in the
Despite decades of research, no completely satisfactory
world(Szinicz2005).Duetothereadyavailability,easeofpro-
explanation of the toxic mechanism and the associated patho-
duction and storage, as well as persistence and stability, SM
physiological processes exists. As a result, there is currently
also represents a potential terrorist agent that poses a signifi-
noeffectivetherapyforrespiratorylesionscausedbySMexpo-
cant threattocivilians aswell.
sure. Nonetheless, it is generally believed that SM acts as an
ExposuretoSMcausesblisteringoftheskinaswellasdam-
electrophile that alkylates cellular and extracellular compo-
age to the eyes and the respiratory tract. Respiratory damage
nents of living tissue such as DNA, RNA, proteins, and other
due to SM inhalation has been found to be dose dependent
biomolecules. As a result, complex cellular events develop,
(Eisenmenger et al. 1991; Papirmeister et al. 1991). At low
to moderate doses, the upper respiratory tract is mostly includingcellcyclearrest,thesynthesisandreleaseofinflam-
affected,whereasathigherdoses,damageisseeninthelower matory mediators, and cytotoxicity. Following these cellular
effects are tissue responses such as inflammation and tissue
damage(Amiretal.2000).Theacuteinjuryisassociatedwith
Addresscorrespondenceto:PrabhatiRay,WalterReedArmyInstituteof arapidandmassivereleaseofdestructiveenzymessuchaspro-
Research—Division of Experimental Therapeutics, 503 Robert Grant Ave.,
teasesandmediatorsofinflammationsuchasproinflammatory
Silver Spring, MD 20910, United States; e-mail address: prabhati.ray@us
cytokines, nitric oxide, and so on (Cowan and Broomfield
.army.mil
Theauthor(s)declarednopotentialconflictsofinterestswithrespecttothe 1993;Smithetal.1995;Cowanetal.2003;Keheetal.2009).
authorshipand/orpublicationofthisarticle. Macrolidesareagroupofantibioticsthatwereinitiallydis-
This work was supported by the Defense Threat Reduction Agency coveredfortheirantibacterialproperties.Thenamemacrolide
(DTRA) Project No. 3.F0003_05_WR_C. The opinions or assertions
is derived from their structure, which is characterized by a
contained herein are the private views of the authors and are not to be
macrocyclic lactone ring with various amino sugars attached
construed as official or as reflecting true views of the U.S. Army or the
DepartmentofDefense. (Bryskier et al. 1993). In recent years, an increasing number
1056
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2011 2. REPORT TYPE 00-00-2011 to 00-00-2011
4. TITLE AND SUBTITLE 5a. CONTRACT NUMBER
Pathological Studies on the Protective Effect of a Macrolide Antibiotic,
5b. GRANT NUMBER
Roxithromycin, against Sulfur Mustard Inhalation Toxicity in a Rat
Model 5c. PROGRAM ELEMENT NUMBER
6. AUTHOR(S) 5d. PROJECT NUMBER
5e. TASK NUMBER
5f. WORK UNIT NUMBER
7. PERFORMING ORGANIZATION NAME(S) AND ADDRESS(ES) 8. PERFORMING ORGANIZATION
Walter Reed Army Institute of Research,Division of Experimental REPORT NUMBER
Therapeutics,503 Robert Grant Ave,Silver Spring,MD,20910
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11. SPONSOR/MONITOR’S REPORT
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12. DISTRIBUTION/AVAILABILITY STATEMENT
Approved for public release; distribution unlimited
13. SUPPLEMENTARY NOTES
14. ABSTRACT
Macrolide antibiotics have been shown to protect airway epithelial cells and macrophages from sulfur
mustard (SM)?induced cytotoxicity. In the current study, the efficacy of roxithromycin in ameliorating
SM-induced respiratory injury was further evaluated in a rat model. Anesthetized rats (N ? 8/group) were
intratracheally exposed to SM by vapor inhalation. For the drug treatment groups, rats were orally given
10, 20, or 40 mg/kg roxithromycin one hr prior to exposure and every twenty-four hr thereafter. After one,
three, or seven days of treatment, sections of the lung were examined and scored for histopathological
parameters. Treatment with roxithromycin ameliorated many of the symptoms caused by SM in some
animals. In particular, treatment at 40 mg/kg for three days showed significant improvements (p < .05)
over the untreated group. When the evaluation was focused on trachea, treatment with roxithromycin for
three days showed a trend of dose-dependent protection; moreover, the groups treated with 20 or 40 mg/kg
of roxithromycin were statistically different (p < .001 and p < .05, respectively) from the untreated group.
These results suggest that roxithromycin protects against some damages associated with SM injury in the
lung, particularly in the upper respiratory tract.
15. SUBJECT TERMS
16. SECURITY CLASSIFICATION OF: 17. LIMITATION OF 18. NUMBER 19a. NAME OF
ABSTRACT OF PAGES RESPONSIBLE PERSON
a. REPORT b. ABSTRACT c. THIS PAGE Same as 9
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Standard Form 298 (Rev. 8-98)
Prescribed by ANSI Std Z39-18
Vol.39,No.7,2011 ROXITHROMYCINPROTECTSAGAINSTSULFURMUSTARD 1057
of studies have suggested that macrolide antibiotics exhibit theSMinethanolwascompletelyvaporizedandinhaled.This
anti-inflammatory functionalities by modulating the produc- passiveexposuresystemincludesaninletone-wayrespiratory
tion of proinflammatory cytokines (Rubin 2004). Although checkvalve fromHansRudolph (KansasCity,MO)toensure
the mechanisms underlying this effect are still unclear, that the only source of air for the animal during the exposure
macrolideshavebeenshowntoaffectseveralpathwaysofthe was through the vapor generator. Exhaled air passes through
inflammatoryprocess,includingthemigrationofneutrophils, a two-way non-rebreathing Rudolph valve and then through a
theoxidativeburstinphagocytes,andtheproductionofproin- charcoal-filtered bleach trap to decontaminate any exhaled
flammatory cytokines (Konno et al. 1994). Our previous in SM.Attheconclusionofthefifty-minexposure,theratswere
vitro studies have demonstrated that macrolide antibiotics disconnected from the vapor generator, the endotracheal tube
protect airway epithelial cells and macrophages from SM- was removed,andthe ratswere returnedtotheir cage.
inducedcytotoxicitythroughthesuppressionofinflammatory
responses(Gaoetal.2007,2008,2010).Theeffectivenessof Roxithromycin Treatment
themacrolideantibioticsinourinvitrostudiespromptedusto
Based on treatment time (duration from the start of the
furthertesttheminaninvivomodel.Inthecurrentstudy,we
treatment to the time of sacrifice), the experiments were
tested the efficacy of a common clinically used macrolide
divided into three major time groups: one day, three days,
antibiotic, roxithromycin (Young, Gonzalez, and Sorkin
andsevendays.Ineachtimegroup,ratswereassignedtoone
1989; Markham and Faulds 1994), in ameliorating SM-
of five treatment groups (N ¼ 8/group): ethanol-exposed/no
induced respiratory injury in a rat model.
drug treatment (control); SM-exposed/no drug treatment
(SM); SM-exposed/treatment with 10 mg/kg roxithromycin
MATERIALS AND METHOD
(RXM 10); SM-exposed/treatment with 20 mg/kg roxithro-
Reagents mycin (RXM 20); SM-exposed/treatment with 40 mg/kg rox-
ithromycin (RXM 40). Inall, 120 rats were used in the study.
Sulfur mustard (2,2’-dichlorodiethyl sulfide; 4 mM)
Roxithromycin powder was suspended in saline by vigorous
was acquired from the U.S. Army Edgewood Research,
mixing and administered p.o. (10 ml/kg) at doses of 10, 20,
Development and Engineering Center (Aberdeen Proving
or 40 mg/kg in respective groups of rats one hr prior to
Ground, MD). Roxithromycin was obtained from Sigma (St.
SMexposure andeverytwenty-fourhrthereafter. Thecontrol
Louis, MO).
group and the SM group receivedan equal volume (10 ml/kg)
of vehicle.
Animals
Male rats (Crl:CD SD BR), weighing 230 to 260 g, were
Histopathologyofthe Lungs
purchased from Charles River Laboratories (Wilmington,
MA) and maintained in an animal care facility that is fully Animals were sacrificed at one day, three days, or seven
accredited by the Association for Assessment and Accredita- days of roxithromycin treatment. After euthanasia, the lungs
tionofLaboratoryAnimalCareInternational.The‘‘Guidefor wereinflatedwithneutralbufferedformalin,fixed,processed,
theCareandUseofLaboratoryAnimals’’(1996)wasfollowed and stained with hematoxylin and eosin. Six sections of lung
during theconductionof theresearch described inthisreport. were taken from each rat, including right cranial lobe, right
medial lobe, right caudal lobe, left lung, right accessory lobe,
SMExposure andtrachea.Eachsectionwasexaminedandscoredonthefol-
lowingparameters: bronchial/bronchiolar exudates,bronchial/
Theexposuremodelhasbeendescribedpreviously(Ander-
bronchiolar epithelial necrosis, bronchial/bronchiolar neutro-
son et al. 1996, 2000). Briefly, rats were anesthetized with an
phil infiltrates, bronchial associated lymphoid tissue (BALT)
intramuscularly (i.m.) administered combination of ketamine
necrosis,alveolarexudate/edema,alveolarhemorrhage,alveo-
(80mg/kg)andxylazine(10mg/kg)andintubatedwithamod-
lar neutrophil infiltrates, alveolar epithelial necrosis, perivas-
ifiedglassPasteurpipette(ca.5cmlong)usingalaryngoscope
cular fibrin/edema, and increase in alveolar macrophages.
to visualize the larynx and a piece of PE90 tubing as a guide Each parameter was given a score from 0 to 3: 0 ¼ normal;
tube. The length of the glass endotracheal tube was sufficient 1 ¼ minimal, present in 1–10% of the section; 2 ¼ moderate,
fordistalplacementinthetracheaatapointbetweenthelarynx present in 10–50% of the section; 3 ¼ severe, present in
and the bifurcation of the trachea. Once in place, the endotra- >50%ofthe section.
cheal tube was secured by wrapping a piece of porous tape
aroundthetubeandtherostrumoftherat.Aglassendotracheal
HistopathologyofTrachea
tube was necessary to minimize absorption of SM. SM (0.25
mg)inabsoluteethanol(100ml)orethanolalonewereplaced The trachea section from each animal was examined and
inawater-jacketed(37(cid:2)C)glassvaporgeneratorcustomfabri- scoredonthefollowingparameters:trachealexudates,tracheal
cated by Atmar Glass (Kennett Square, PA). Spontaneously neutrophil infiltrates, tracheal epithelial attenuation, and tra-
breathing rats were connected to these devices and exposed cheal epithelial necrosis or loss. Each parameter was given a
accordingly for fifty min. By the end of the exposure period, score from 0 to 3: 0 ¼ normal; 1 ¼ minimal, present in
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1058 GAOETAL. TOXICOLOGICPATHOLOGY
..
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E
FIGURE1.—Histopathologicalsectionsshowingtypicalchangesseeninsulfurmustard(SM)–exposedanimalsinthetracheaarea.Innormalani-
mals,thetrachealumenwasfreefromexudateanddebris(A,2(cid:3)),andtherespiratoryepitheliumwasintactwithanormalpseudostratifiedcolum-
narmorphology,andnoinflammatorycellswerepresent(B,20(cid:3)).InSM-exposedanimals,thetracheallumenwasfilledwithexudates(C,2(cid:3)),
whichwerecomposedoffibrin,necroticdebris,andinflammatorycells(D,20(cid:3)).Mostoftherespiratoryepitheliumwaslost,andtheremaining
epithelialcellsweremarkedlyattenuated(E,20(cid:3)),withthenecroticepithelialcellsscatteredalongthesurface(F,40(cid:3)).
Downloaded from tpx.sagepub.com at USAMRIID on December 1, 2011
+
GroupaverageSDperanimal +0.710.76+0.500.76+1.001.73 +19.6310.39+33.1712.95+10.335.51 +20.5711.70+23.139.20+16.752.99 +22.291.80+27.507.58+14.331.15 +16.507.18+15.005.74+18.004.47
s
arosi
Alveolstiocyt 0.000.030.04 0.200.470.13 0.290.500.20 0.260.400.20 0.200.000.40 group.
hi ch
a
e
a Perivascularfibrin/edema 0.030.050.12 0.680.570.13 0.310.300.05 0.260.670.00 0.230.200.00 alsoshownfor
ers. D)is
alparamet Alveolarepithelialnecrosis 0.000.000.00 0.030.070.00 0.000.000.00 0.000.030.00 0.000.040.00 deviation(S
c d
ogi ndar
orallpathol Alveolarneutrophilinfiltrates 0.090.030.00 0.180.330.13 0.140.250.10 0.200.500.07 0.100.240.56 group.Thesta
f by
entalgroup Alveolarhemorrhage 0.000.000.00 0.030.030.00 0.060.000.00 0.170.000.00 0.070.000.00 wereaveraged
m
achexperi Alveolarexudate/edema 0.000.000.00 0.380.230.07 0.200.200.20 0.200.300.13 0.100.120.08 mycin.hetotalscores
ne hroent
animali BALTnecrosis 0.000.000.00 0.080.000.00 0.000.000.00 0.230.000.00 0.070.040.00 ¼XMroxitanimal.Th
er Rch
veragescorep Bronchialneutrophilinfiltrates 0.000.000.03 0.311.080.33 0.500.670.54 0.570.750.61 0.390.400.43 ¼sulfurmustard;eresummedforea
A Mw
— Sn
T1.ABLE Bronchialepithelialnecrosis 0.000.000.00 0.771.640.44 1.001.060.92 0.901.330.61 0.781.000.72 mphoidtissue;andeachsectio
lyer
Bronchial/Treatmentbronchiolartime(days)exudate 10.0230.0070.00 10.9031.3970.56 11.1031.0870.88 11.1430.9270.83 10.9430.6771.00 ¼ations:BALTbronchialassociatedlastcolumn,scoresforeachparamet
Treatmenttype Control SM RXM10 RXM20 RXM40 AbbreviaForthe
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1060 GAOETAL. TOXICOLOGICPATHOLOGY
1–10% of the section; 2 ¼ moderate, present in 10–50% of
the section; 3 ¼ severe, present in >50% of the section. In
some cases, as tracheal epithelium was injured and individual
cells were lost, the remaining cells took on a flattened or
attenuated appearance, thus covering the same surface area
with fewer cells. In order to account for these changes, both
necrosis/loss and epithelial attenuation were scored and the
average was used. In cases where there was no epithelium
left, the score for the necrosis/loss was used as the average
of the two.
DataAnalysis
Dataareshownasmean+standarddeviation.Groupcom-
parisonswereconductedusingone-wayANOVAfollowedby
Tukey’s post hoc multiple comparison test. Pathology data
wereanalyzedacrossgroupswithachi-squaretest.Forsignif-
icance analysis, all groups treated with roxithromycin were
compared to the group exposed to SM only using a Fisher’s
exact test. Significant results were identified when p < .05 or
smaller.
RESULTS
Overall Histopathological Changes
The most profound histopathological changes in the SM-
exposed animals were found in the trachea, bronchi, and
bronchioles. These changes consisted of exudates, epithelial
necrosis and loss, and neutrophilic infiltrates. In trachea, exu-
dates composed of sloughed cells, fibrin, necrotic debris, and
inflammatorycellsoftenfilledthelumen.Trachealepithelium
was often necrotic or lost. Remaining epithelial cells were
markedlyattenuated, andepithelialattenuationincreased over
time.Oftenneutrophilswerepresentinthemucosaandlamina
propria. Figure 1 highlights some histopathological sections
with typical changes seen in SM-exposed animals in the tra-
chea area. Similar to the trachea, the lumen in bronchi and
bronchioles was often filled with sloughed cells, fibrin,
necrotic debris and inflammatory cells. Epithelium was fre-
quentlynecrotic,attenuated,orlost,andattenuationincreased
over time. Neutrophilic infiltrates were often present in the
mucosa and lamina propria. Some of these changes are
shown in Figure 2.
Consistent with previous in vivo studies using this model
(Andersonetal.1996),changesinthealveoliwerenotaspro-
minent as they were in the trachea, bronchi, and bronchioles.
The most common and profound changes were the filling of
alveolarspaceswithedemaandfibrinoranincreasednumber
ofalveolarmacrophages(Figure2).Lesscommonly,therewas
FIGURE 2.—Histopathological sections showing typical changes seen Figure2(continued).bronchiole,andtheabsenceofcellsanddebris
in sulfur mustard (SM)–exposed animals in bronchiole and alveolar inthelumen.(B)SM-exposedanimals(40(cid:3)).Thealveolarseptawere
areas. (A) Normal (control) animals (20(cid:3)). The open arrow points markedly thickened by inflammatory cells and edema. (C) SM-
tothealveoli.Notethethicknessoftheseptaandtheabsenceofdebris exposedanimals(10(cid:3)).Thebronchiolewallwasmarkedlyexpanded
orcellswithinalveolarspaces.Theclosedarrowindicatesbronchiole. byinflammatorycells,therespiratoryepitheliumwascompletelylost,
Notetheuniformpseudostratifiedcolumnarepithelialcellsliningthe andthelumenwasfilledwithinflammatorycells,edema,anddebris.
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Vol.39,No.7,2011 ROXITHROMYCINPROTECTSAGAINSTSULFURMUSTARD 1061
Table 1 shows group averages for each parameter used in
the pathological evaluation. The trachea was included in the
columnsforbronchialepithelialnecrosisandbronchialneutro-
phil infiltrates. SM exposure resulted in higher scores in all
areas, but with only minimal differences in BALT necrosis,
alveolar hemorrhage, and alveolar epithelial necrosis. It was
noticed that lesions were more consistent and more severe in
larger airways (trachea, bronchi, and bronchioles) than they
were in alveoli. Lesions in these larger conducting airways
increased inseverity, on average between days oneand three.
Bydayseven,thelesionsinthelargerairwayswerelesssevere
than day three. Lesions were not severe in all sections of the
lungs in many of the animals, but all lobes were affected in
someanimals.
Similar to the SM group, the groups treated with roxithro-
mycinalsohadhigherscoresinthelargerconductingairways
whencomparedtothealveoli.Thetwogroupstreatedwith10
or 20 mg/kg roxithromycin also had a pattern of increased
severity on day three when compared to days one and seven.
The magnitude ofdifference appeared to decrease asthe dose
increaseduntilatthehighestdose(40mg/kg)therewasnodif-
ference betweendays one,three, andseven.
Thegroupaveragetotalscore(lastcolumninTable1,which
isthesumofallparametersandallsectionsaveragedbygroup)
isgraphically displayed inFigure3, divided into three groups
based on treatment time. In most cases, there were no signifi-
cant differences between animals treated with roxithromycin
and their respective untreated counterparts (exposed to SM
only). However, for the three-day groups, there was a signifi-
cant difference between the SM-exposed animals and those
treated with 40 mg/kg of roxithromycin (p < .05), suggesting
the effectiveness of the drug at the current dose and duration
oftreatment.
Histopathologyon TracheaOnly
Because the most consistent and most severe lesions were
found in larger airways, the trachea was evaluated separately
fromtherestofthelung.Thetracheasectionfromeachanimal
wasexaminedandscoredonthefollowingparameters:tracheal
exudates, tracheal neutrophil infiltrates, tracheal epithelial
attenuation, and tracheal epithelial necrosis or loss. Table 2
shows the averages of histopathological scores from all treat-
ment groups. The trends are similar to those seen in the com-
FIGURE 3.—Graphical representation of group average total score of plete set of data (Table 1). In the SM-exposed groups as well
histopathology arranged by treatment time. (A) One-day treatment asthegroupstreatedwith10or20mg/kgofroxithromycin,the
group.(B)Three-daytreatmentgroup.(C)Seven-daytreatmentgroup. severity was highest on day three. However, for the groups
Thegroupaveragetotalscoreswerecalculatedbysummingthescores treatedwith40mg/kgofroxithromycin,lesionsinthetrachea
ofallparametersandallsectionsforeachanimal(animaltotalscore)
increased inseverity with time.
and then averaging the animal total scores by group. Comparisons
Thegroupaveragescore(sumofallparametersforeachani-
were made between animals treated with different concentrations of
malaveragedbygroup)showninthelastcolumnofTable2is
roxithromycinversustheirrespectiveuntreatedcounterpartsexposed
graphically displayed in Figure 4, divided into three groups
tosulfurmustardonly(SM).*p<.05.
based on treatment time. Treatment with roxithromycin for
necrosis of alveolar epithelium or neutrophilic infiltrates into threedaysshowedatrendofdose-dependentimprovement,and
the alveolar spaces. Frequently in SM-exposed animals, the thegroupstreatedwith20or40mg/kgofroxithromycinwere
perivascular spaces were expanded byedema andfibrin. statisticallydifferent(p<.001andp<.05,respectively)from
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1062 GAOETAL. TOXICOLOGICPATHOLOGY
TABLE2.—Averagescoreperanimalineachexperimentalgroupforpathologicalparametersontracheaonly.a
Treatment Treatmenttime Tracheal Trachealneutrophil Trachealepithelial Trachealnecrosisor Groupaverage+SDper
type (days) exudate infiltrates attenuation loss animal
Control 1 0.00 0.00 0.00 0.00 0.00+0.00
3 0.00 0.00 0.13 0.00 0.13+0.35
7 0.00 0.00 0.00 0.00 0.00+0.00
SM 1 1.38 0.63 1.88 1.75 5.63+2.39
3 2.50 2.33 3.00 3.00 10.83+1.17
7 1.00 1.00 3.00 1.67 6.67+2.08
RXM10 1 2.14 0.71 2.14 2.43 7.43+2.70
3 2.38 1.38 3.00 2.88 9.63+1.19
7 1.00 1.00 2.75 2.25 7.00+3.37
RXM20 1 1.86 1.14 2.29 2.43 7.71+3.20
3 1.33 1.17 2.67 3.00 8.17+0.75
7 0.33 0.67 2.00 2.00 5.00+3.46
RXM40 1 1.83 0.67 2.17 2.00 6.67+1.97
3 1.60 1.20 3.00 2.60 8.40+2.07
7 2.00 1.40 3.00 2.80 9.20+0.45
Abbreviations:SM¼sulfurmustard;RXM¼roxithromycin.
aForthelastcolumn,scoresforeachparameterweresummedforeachanimal.Thenthetotalscoreswereaveragedbygroup.Thestandarddeviation(SD)isalsoshownforeachgroup.
the untreated group (Figure 4), suggesting that treatment with roxithromycin, a representative macrolide antibiotic, for its
roxithromycinreducessomeofthedamagecausedbySMinha- efficacyagainstSM-inducedlunginjuryusingaratSMinhala-
lation inthe trachea. tionmodelthathasbeenpublishedpreviously(Andersonetal.
1996,2000).
Surviving AnimalCounts The immunomodulatory effects of macrolides have been
observed in clinical studies of chronic inflammatory airway
The number of surviving animals at the end of the experi-
diseases (e.g., DPB) after long-term treatment with low dose
mentineachgroupislistedinTable3.Ingeneral,there were
ofmacrolides(Rubin2004).Forroxithromycin,adailydosage
somegainsinthegroupstreatedwithroxithromycinexceptfor
ofonetablet(150mg)wasusuallyadministeredforaperiodof
those treated with a dose of 10 mg/kg, where the number
two to three months (Kimura et al. 1997; Suzuki et al. 1997,
remained the samefor allgroups exposedtoSM. Inallcases,
2000). The dosage is equivalent to 1.5 to 3.0 mg/kg, using a
theanimalsdiedwithinthreedaysafterSMexposure;however,
body weight for an average patient in the range of 50 to 100
compared with animals exposed to SM only, treatment with
kg.However,thistreatmentregimencannotbeadopteddirectly
roxithromycinnotonlyincreasedthenumberofsurvivors,but
inthecurrentstudyas,inthecaseofSMinhalationexposure,
alsodelayedthedyingoftheanimalstosomeextent(Figure5).
airwayinflammation isnotchronicbutrather acute.Insevere
cases, symptoms develop within hours after exposure (Eisen-
DISCUSSION
mengeretal.1991;Papirmeisteretal.1991).Assumingabal-
SMinhalationleadstorespiratorydamagecharacterizedby ancebetweendrugdosageandtreatmentdurationmayexist,in
quick onset of inflammatory responses and subsequent pul- thepresentstudywetestedroxithromycinusingrelativelyhigh
monary dysfunction. The clinical signs of SM inhalation doses(10,20,and40mg/kg)forshortertreatmenttimes(one,
include asthma, chronic bronchitis, bronchiectasis, and pul- three, andseven days).
monaryfibrosis(EmadandRezaian1997).Macrolideantibio- The best results were obtained for the three-day treatment
ticshaveshowneffectivenessinavarietyofchronicrespiratory groups with doses of 20 mg/kg and 40 mg/kg, respectively
diseasessuchasdiffusepanbronchiolitis(DPB),asthma,cystic (Figs. 3B and 4B). Clearly, treatment for one day is too short
fibrosis,chronicbronchitis,andchronicsinusitis(Rubin2004). foranytherapeuticeffectofthedrugtobeseen(Figs.3Aand
Althoughtheunderlyingmechanismsarestillunclear,itisnow 4A). We expected to see more prominent beneficial effect for
generally believed that the effectiveness of macrolides is a the seven-day treatment groups. On the contrary, compared
result of their immunomodulatory function. Macrolides have to the SM-exposed group, a higher pathological score was
been shown to affect several pathways of the inflammatory obtainedforthegrouptreatedwith40mg/kgofroxithromycin
process, including the migration of neutrophils, the oxidative (Fig. 4C). This suggests that longer treatment (seven days) at
burst in phagocytes, and the production of proinflammatory highdose(40mg/kg)mayhaveadverseeffectonpathological
cytokines (Konno et al. 1994). Our previous in vitro studies outcome of respiratory injuries caused by SM exposure. The
have demonstrated that macrolide antibiotics protect airway doses used in the current study (10–40 mg/kg) are ca. ten to
epithelial cells and macrophages from SM-induced cytotoxi- twenty times higher than those used in clinical studies (1.5–
city through the suppression of inflammatory responses (Gao 3.0 mg/kg). Although macrolides are considered as well-
etal.2007,2008,2010).Inthecurrentstudywehaveevaluated tolerated antibiotics, adverse side effects and toxicity of
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Vol.39,No.7,2011 ROXITHROMYCINPROTECTSAGAINSTSULFURMUSTARD 1063
TABLE3.—Numberofsurvivinganimalsineachexperimentalgroup.a
Treatmenttime(days) Control SM RXM10 RXM20 RXM40
1 8 7 7 7 7
3 8 6 7 7 6
7 8 3 5 3 5
Abbreviations:SM¼sulfurmustard;RXM¼roxithromycin.
aEachgrouphadeightanimals(N¼8)atthebeginningofexperiment.
FIGURE5.—Numberofsurvivinganimalsateachdayineachgroupin
theseven-daydrugtreatmentgroups.
patients on the tolerability of clinically available macrolide
antibioticsrevealedthatgastrointestinalreactionsrepresentthe
most frequent disturbance due to induction of endogenous
release of motilin (Periti et al. 1993). Incidences of adverse
reactions in the heart, liver, central nervous system, and
immunesystem,althoughrare,havealsobeenreported(Periti
et al. 1993). The findings of the current study may suggest a
possibleadverseeffectofmacrolideantibiotics,whenadminis-
teredathighdosesandforlongperiodsoftime,ontherespira-
torysystem.
The dose of SM used in the exposure (1.0 mg/kg) also
appeared to be too high for the experiment. For the seven-
day experiment, more than 50% of the animals died within
three days after SM exposure in some groups (Figure 5),
namely,lessthan50%oftheanimalssurvivedtheexperiment
(Table 3). The number of surviving animals in these groups
FIGURE4.—Graphicalrepresentationofgroupaveragescoreofpathologi-
maybetoolowtoproducemeaningfulresultsfrompathologi-
cal parameters focusing on trachea, arranged by treatment time. (A)
One-daytreatmentgroup.(B)Three-daytreatmentgroup.(C)Seven-day cal evaluations(Fig.3C).
treatmentgroup.Thescoreswerecalculatedbysummingthescoresofall Aspreviouslyreported,theSMexposuremodelusedinthe
parametersforeachanimal(animaltotalscore)andthenaveragingtheani- current study did not produce even exposure along the entire
maltotalscoresbygroup.Comparisonsweremadebetweenanimalstreated respiratory system (Anderson et al. 1996). More severe dam-
withdifferentconcentrationsofroxithromycinversustheiruntreatedcoun- ageswereseenintheupperrespiratorytract(trachea,bronchi,
terpartsexposedtosulfurmustardonly(SM).*p<.05.***p<.001. and bronchioles) than in the deeper lung (alveoli). This may
partlyexplaintheresultthatmoreprominenteffectofroxithro-
macrolide antibiotics have been reported in many cases (Jain mycinwasseenwhenhistopathologicalevaluationwasfocused
and Danziger 2004; Periti et al. 1993). Clinical data over a on trachea only instead of on the entire respiratory system
forty-year period in many thousands of adult and pediatric (Figs.4B vs.3B).
Downloaded from tpx.sagepub.com at USAMRIID on December 1, 2011
1064 GAOETAL. TOXICOLOGICPATHOLOGY
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