IJIIC FILE COPY jIJIIC FILE COPY j .SHORT-TERM IN VITRO SCREENING STUDIES RELATED TO THE INHALATION TOXICOLOGY OF POTENTIALLY TOXIC AEROSOLS FINAL REPORT Blaine C. …

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IJIIC FILE COPY j .

SHORT-TERM IN VITRO SCREENING STUDIES RELATED TO THE INHALATION

TOXICOLOGY OF POTENTIALLY TOXIC AEROSOLS

FINAL REPORT

Blaine C. Gross, Ph.D.00 Judith A. Graham, Ph.D.

August 197

Army Project Order 84PP4850

Touicolop Branch

Inhalation Touicolog Division D Tr CU.S. Environmental Protection Agency SEP 16 87

Research Triangle Park, NC 27711

U.S. ARMY MEDICAL RESEARCH AND DEVELOPMENT COMMAND

Fort Detriek, Frederick, MD 21701-5012

Project Officer. Gunda Reddy, Ph.D.

Health Effects Research Division

U.S. ARMY MEDICAL BIOENGINEERING RESEARCH AND DEVELOPMENT LABORATORY

Fort Detrick, Frederick, MD 21701-5010

Approved for public release;

distribution unlimited

The findings in this report are not to be construed as an officialDepartment of the Army position unless so designatedby other authorized documents.

7 09 15 10

, .OTICE

This paper has been revleuad by thp Health Effects Research Ukabortry, U S& Environmental. trotetion Agency, and approved for publication. Mention of trade namse ormweial produc's

h m bot constitute endorsement or recommendation for use.

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SG-URrVTY CLASSIFICATION OF•HS A &AGEREPORT DOCUMENTATION PAGE M Ap..•. e,

• ~~~0"10 NO.0;O-lt

IS. REPOXT SECURIT," CLASSIFICAT;ON Ib. RESTRICTIVE MARKINGSUnclasesified24. SECURITY CLASSIFICATION AUTHORIT•V 3. DiSTR'UUTIONIAVAILABILITY OF REPORT

2b. OECLASSIFICATIONIDOWNGRAOING SCI.EDULi. Approved for public release;distribution unlimited

4. PERFORMING ORGANIZATION REPORT NUMIER(S) S. MONITORING ORGANIZATION REPORT NUMBER(S),

6., N E OF PqfFORMINO O ORG TN 66. OFFICE SYMBOL. 7a. NAME OF MONITORING ORGANIZATION

E ffects Research Laborarory ...... U.S. Army Medical Bioengineering Research

t.S. Environmental Protection Agency Develoomen. Laborator .[ C. ADDRESS (CtySe,.aZPCo) 7b. ADDRESS (City, State, am@ ZIP Cow.)

Researzh Triangle Park, NC 27711 Fort Detrick, Frederick, MD 21701-5010

I1. NAME OF FUNOING, SPONSORING ISO. OFFICE SYMBOL 9. PROCUREMENT INSTRUMENT IDENTIFICATION NUMBERORGANIZATION U. S. Army Medical j(Nf e&ipeb')

'esearch & Development Couniand SGRD-RMI-S 84PP4850SC ADDRESS (Oty, Staet, and ZIP Cad#) Ito 10. SOURCE OF FUNDING NUMBERS

PROGRAM PRO4ECT TASK. WORK .j NIT

Fort Detrick, Frederick, .M 21701-501.2 ELEMENT NO. NO. CCESSION NO.

11 ITL (include Socurity Oamficartonj

Short-Term In Vitro Screening Studies Related to the Inhalation Toxicology of Potentially:oxic Aerosols,.PERSONAL. AUTI40R(S)

Elaine C. Grose, Ph.D. and Judith A. Graham, Ph.D.3•.. T'•'PE OF REPORT 13b. TIME COVERED 114. DATE OF REPORT (Yee•r,•tof•twy) iS. PAGE COUNT

Final FROM June 84 To Apr 86 1987 August 2316. SUPPLEMENTARY tNOTATlON

7 COSATI CODES 18. SUBJECT TERMS (Coftn rvere of necsuty dna .t...y .y IxoCt numoeri

;EL GROUP I SUE-GROUP acid phosphatase, graphite, adenosine triphosphate.alveolar macrophages, brass, bactericidal, activity,tracheal organ culture

9aBSRACT (Continue on reverie it teceswy &ad idenifpmyy bo ock numeer)SThe toxicity-of jCE001 particles was tested using the rabbit alveolor macrophage (RA.,!)

test, hamster tracheal .-rs,,-azaa ultures- and in vivo pulmonary6actericidal actovttb in mice..tR

In the RAM test, incubation of alveolar macrophage.J(AM) with the JCE001 particlesdecreased - O..5) viability, cellular adenosine 5,y-triphosphate (AT?) and totalprotein levels. The lowest effective dose as determined by Williams' test was 30 ,g/•r.of JCE001 particles for all parameters examined. The presence of soluble cytotoxiccomponents was established. The nature and rate of release of soluble toxic comgonentsfrom the JCE001 particles were important contributing factors in the mechanism by whichthese particles produced their toxic effects.

(Cont.) >0 ISTRIBUTION/AVAILABIUTY OF ABSTRACT 21. ABSTRACT SECURITY CLASSIFICATION

:-3 UNCLASSIFPEDO/JNUMITED Cfl SAME AS CP'r 0 :1r'C USERS Unclassified2,1a. '4AME OF RESPONSIBLE NOIVIOUAL , 22b. TELEP4ONE (IUnclle Area Cooe) 2,c. OFPCE SYN1BOL

S Virzinia Miller 301-663-7"325 SGRD-.MI-SO0 Form 1473. JUN 86 P'rv,ouse n sOMareomo 6 re. >ECUR17v CLASSIFICA• TON OF T-615 'Qj_

19. (Cont.)

e7 single 1-hr in vitro exposure of hamster tracheal ring organ cultures to theJCE001 particles was conducted to determine the dose response on ciliated respiratoryepithelium. JCE001 particles -caused a significant decrease in cilia beating frequencyand an increase in cytopathological alterations..when tasted at 250 .ig/mL and above.

-Morphological alterzaons in ý the trachea ep thelium were observed with light microscopyand scanning electron micrO3COPy. An inhibitory effect of JCE001 partl'cies on RNAsynthesis, pro .i esis, and succinic dehydrogenase activity was not demonstrated.Explanta.-Teated with 1000 ig/ImL of JCEO01 particles showed an inhibition of DNAs yIiesis.

SThe effect of the JCEO0l particles on the ability of AM to kill inhald F-pneumoniae %as.ýj.s asued..) No statistical Main effect of treatment was observedalthough thi~bactericidal activity of all groups treated with ;he JCE001 particleswas markedly reduced compared to that of controls. -

In addition, the effects of the Asbury #650 and Dixon KS-2 graphite particles onthe putmonary bactericidal activity of mice to inhaled E35 S]K. pneumoniae were measured.,Significantly decreased bactericidal activities were observed in the 250 pg-treatmentgroups compared to both naive and vehicle controls. Intratracheal instillation ofAsbury #650 and Dixon KS-2 particles did not affect the pulmonary bactericidal activity.

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FOREWORD

In Anducting research using animals, the investigator(s) adhered to the "Guide for the Care andUse of Laboratory Animals," prepared by the Committee on Care and Use of Laboratory Animals ofthe Institute of Laboratory Animal Resources, National Research Council (NIH PublicationNo. 86-23, Revised 1985).

Citations of commercial organizations and trade names in thin report do not constitute anofficial Department of the Army endorsement or approval of the products or services of theseorganizations.

DTIC ,TAB , 13Unannounced •Justification... . .

By ...... '............- _..,

II

ACKNOWLEDGMENT

The authors would like to acknowledge the participation of the following people in the study:Catherine Aranyi, Jeannie Bradof, Dorothy Davis, Charles Gradle, Kathy Kettles, Robert Lange,William Mega, William O'Shea, Glenn Pasek, Jnhnnie Raymond, Josephine Reed, Leonard J. Schiff,Ph.D., and Robert Sherwood, Ph.D. (UIT Research Institute).

TABL9 OF CONTENT

FOREWORD -------------------------------------------------------ACK±NOWLEDGMENTS --------------.r.-.------------ ................ 2LIST OF FIGURES ..---------------------- 5LIST OF TABLES ---

INTRODUCTION ------------------------------------- -------------------- 7

PART 1. THE IN VITRO EFFECTS OF JCEOO PARTICLES IN THE RABBITALVEOLAR MACROPHAGE TEST ---------------------------------- 7

Objective ---.-- ----------.-------------------------------------- - 7,Materials and Methods .......---------------------------------------- 7

Test Particles .--------------------------------------------------- 7The RAM Test .......... ---------------------------------. 7Soluble Cytotoxic Components ------------------------ -------------- 8Statistical Analysis -.----- w------------------------------------- 8Experimental Design ------ w ......... - ---------- -;- 8

Results - ----.................------------- = ------------------- 8Tost Particles.---., -------------------- ------------------------.-- 8Soluble Cytotoxic Components--------------------------------- 8Cytotoricity of Particles .-----------------------------------------. 9

Summary ----------------------------------------- ---------------- 9

PART 2. THE EFFECTS OF JCE00I PARTICLES ON PULMONARYBACTERICIDAL ACTIVITY ---------------------------------------- 9

Objective ---------------------------------- ----------------------- 9Materials and Methods -----------------------------------------------. 9

Test Particles ------------------------------------------- . 9Animals ------------------ . ------------------------------------- 11Intratracheal Instillation ----------------------------------------- 1 IPulmonary Bactericidal Activity ------------------------------------. IStatistical Analysis ------------------------------------------ ; ---- 12

Results --------------------------- . -------------------------------- 12Summary --------------- ------------------------------------------ 12

PART 3. THE EFFECTS OF JCE001 PARTICLES IN HAMSTER TRACHEALRING ORGAN CULTURE ASSAYS .---------------------------------- 13

Objective --------------------------------------------------------- 13Materials and Methods ----------------------------------------------- 13

:3

TABLE OF CONTENTS (Cont.)

Results -----------------------------------------------------------. 13Ciliary Activity and Cytopathology ...........................----- 13Histopathology (Light Microscopy) ---------------------------------- 14Scanning Electron Microscopy .-------------------------------------- 14Aut6oradiography --------.-------------------------------------- 18

Tritiated Thymidins Incorporation ------------------------------- 18Tritiated Uridine Incorporation ----------------------------------- 18Tritiated Leucine Incorporation --------------------------------- 18

Succinic Dehydrogenase Activity (Histochemistry) ---------------------- 18Summary ------------------------ --------------------------------- 18

PART 4. THE IN VIVO EFFECTS OF GRAPHITE PARTICLES ASBURY #650AND DIXON KS-2 ON PULMONARY BACTERICIDAL ACTIVITY --------- 18

Objective --------------------------------------------------------- 18Materials and Methods ----------------------------------------------- 18

Test Particles ..-------------------------------------------------- 18Animals ------------------------------------------------------ 19Intratracheal Instillation .---------------------------------------- 19Pulmonary Bactericidal Activity ----------------------------------- 19Statistical Analysis .---------------------------------------------- 19Experimentzl Design -------------------------------------------- 19

Resuts--- .-------------------------------------------------------- 19Summary ---------------- ------------------------------------------- 20

LITERATURE CITED ---------------------------------------------------- 21

4

SI I I I I I• I I •!

LIST OF FIGURES'

IA. Ciliated and Microvillus Cells of Epithelium of Control Trachea Explants, 1100iXl ---. i5

18. Ciliated and Microvillus Cells of Epithelium of Control Trachea Explants, 2200X 15

2A. Particles Recting on Tips of Cilia in JCE001-treated Tracheal Explants, 2200X ------ 16

2B. Particles Resting on Tips of Cilia in JCEOOI-treated Tracheal Explants, I 10OX ------- 16

3A. Particles Attached to Microvillus Surface in JCE001-treated Tracheal Explants,2200X .-------------------------------------------------------- .17

3B. Crater-like Structure in Microvillous Surface ofJCEOO1-treated Tracheal Explants,500X .----------------------------------------------------------- 17

5MA

I Effects of2O-hr In Vitro Exposure at 370C to JCE001 Particles in RAM Test ------ 10

2 Conbined Effect- of 20-h.- In Vitro Exposure at 37*C to JCE001 Particles in

the RAM Test ---------------------------------------------------- 1I

3 Pulmonary Bactericidal Activity of CD- I Mice to Inhaled (35SlK. Pneumonia 20 hr

after Intratracheal Instillation of JCEOO1 Particles ------------------------- 12

4 Mean Cilia-beating Frequency in Hamster Tracheal Ring Organ Cultures Exposed

1 hr to JCE0OOI Particles ----------------------------------- 13

5 Percentage of Normal Epithelium in Hamster Trachcal Ring Organ Cultures after

a 1-hr In Vitro Exposure to JCEOOI Particles ------------------------ 14

8 Pulmonary Bactericidal Activity in CD-I Mice toiInhaled (391KN. Pnuoi 24 hr

after Intratracheal. Instillation of Asbury #650 and Dixon KS-2 Particles -------- 20

JCE001, Asbury #650 and Dixon KS-2 particles were tested to determine their toxicity.In vitro tests, including rabbit alveolar macrophage viability, adenosine 5"triphosphate (ATP)content, total protein, and particle solubility, etc., were performed on rabbit and alveolarmacrophages to determine the specific toxicity of the JCEO01 particles. Ciliary-beating frequencyand histopathology of tracheal organ cultures were also investigated in hamsters with the JCE001particles. Further testing was done on pulmonary bactericidal activity in mice for all particles.

PART 1. THE IN VITRO EF•FECTS OF JCE001 PARTICLES IN THE RABBIT ALVEOLARMACROPHAGE TEST

OBJECTIVEThe rabbit alveolar macrophage (RAP) test was used to determine the effects of JCE001

particles on alveolar macrophage (AM) viability, levels of ATP, and total protein.

MATERIALS AND METHODS

Test Particles

Atlantic brass, Richgold 1800, designated as JCE001 particles, was provided by the U.S. ArmyMedical Research and Development Command and was stored at ambient temperature. According tothe data sheet that accompanied the test particles, the particles had a mass median aerodynamicdiameter (MMAD) of 2.3 pim, with a geometric standard deviation of 1.61. The elemental analysisshowed 65 to 7A% copper, 26 to 35% znc, and traces of lead, arsenic, aluminum, and various fattyacids.

The LIAM Test

Male 2- to 3-kg New Zealand albino rabbits (Hazelton Dutchland L 'oratories, Denvwr, PA)were killed by injection of sodium pentobarbital (75 mg/kg) in the marginal ear vein. AM obtained bytracheobronchial lavage were centrifuged and washed in Hanks' Balanced Salt Solution (HBSS). Cellviability and total and differential cell counts were determined.' An AM suspension and a particlesispension were prepared in Medium 199/HBSS suppiemented with serum and antibiotics at twicethe projected exposure concentrations, and equal volumes of the two suspensions were mixed. Aconstant concentration of 106 AMVmL was maintained in the test suspension, the concentration ot .. eperticles was increased serially from 10 to 1000 pg/mL. The test suspensions were incubateJ in •eilsof disposable plastic ':luster dishes placed on a rocker platform for 20 hr at 37"C in a humidified 5%carbon dioxide (C09) atmasphere.

The test parameters included the percentagc of AM viability, micrograms of protein permilliliter and femtograms of ATP x 106 per microgram of total proteir. Immediately after incubation,the percentage of AM viability was determined by Trypan blue exclusion technique.- The testsuspensions were subsequently washed, centrifuged, and resuspended in HBSS before monitoringtotai protein and ATP levels. An aliquot of the cell suspension was treated with sodium deoxycholate,the rosulting cell lysate centrifuged at 10,000 x g, and the supernatant used for the Lowry proteinassay.' After extraction from the cells with dimethyl sulfoxide (DMSO) taken from another aliquot..ATP was determined using the luciferin-luciferase reaction in a DuPont 760 LuminescenceBiometer.'

S•]wluble CvY-Wtoxjc Components

To determine the extent to which leachable components contribute to the total cytotoxic effect,sampls of the test particles were incubated in the culture medium af. 1)00 pg/nL as described above,omitting AM and serum. After incubation, the particles were removed from the media byultracentrifigation at 10,000 rpm x 15 main, filtered using 0.22 pm flwters (Falcon Co.), andresuspended in fresh medium. Then the filtered supernatant, the resuspended preincubatedparticles, and the origi.-al unseparated particle suspension after preincubation were testedseparately for cytotonicity using only AM viaoility.Statitca Analysis

Total protein values are given as micrograms per 1.0 mL of the initial incubation sample. ATPlevels are expressed in femtograms of ATP per microgram of total protein (experimentallydetermined), or per milliliter of the ir-itial incubation sample. Changes in total protein and AT".levels are Gxpressed as a percentage of the controls. Because viability of the control AM is not affectedby the incubation period, viability values are reprrted directly as a percentage of the total AM in theexperimental sample. One-way analysis of variance (ANOVA) was used to ovaluate the effects of theparticles on the various parameters tested. Williams' test " was used to determine the lowesteffective dose.Experimental Desgn

Because of difficulties in suspending the test particles in the aqueous incubation medium usedfor the assay, a concentrated (10,000 pg/mL) stock suspension was prepared in aqueous mediumcontaining 1% isopropyl alcohol (IPA), and the test concentrations were obtained by diluting thissto.k. n!ased on the results of a range-finding assay, the ,-a-tidles were tested at 10, 50, 250,500, and1000 pg/mL. Three to four culture wells with I06 AM/mL were used at each concentration with AMpooled from two to four rabbits as needed to obtain an adequate number of cells for the assays. Tworeplicate experiments were conducted. The particles were screened for soluble cytotoxic componentsprior to conducting the main RAM test.

RESULTSTest Particies

The JCE001 particles are very hydrophobic and floated on, the aqueous Medium 199, coating thecontainer walls without entering suspension. An appropriate wetting agent had to be used todisperse the particles in the culture medium. Ethyl alcohol, acetone, IPA, glycerine, and DMSO weretested. iPA proved to be the best; a good suspension was obtained, and relatively fiw of the testparticles coated the container walls.

Solubl~e CytotoxicC Cmponentl

Before assessing the in vitro effects of the JCEOOI particles suspended with AM in the liquidmaintenance medium, it was necessary to determine whether any toxicity could have beencontributed by material solubilized from the particles under the expsure conditions. Results wereobtained in examining the test sample for potentially toxic soluble components. The data show thatthe filtered supornatant produced a marked decrease (38%) in AM viability relative to the unexposedcontrols (93.9%). This clearly demonstrates that soluble cytotoxic components were released from the"JCE001 particles during incubation with the cells. The reiuspended preincubated particles remainedcytotoxic (19.2%), as well as the original unseparated particle suspension after proincubation (29%).Thus, the nature and rate of release of soluble toric components from the JCE001 particles wereimportant contributing factors in the mechanism by which these particles produced their toxic effects,

Cytotoxicii• of Particles

The results for the two replicate experiments are summarized in Table 1. The viability valueswe.- recorded as the percentage of total AM in dAch experimental sample, whereas the data for totalprot•in and ATP were expressed as the percpntage of the corresponding control responses.

Tab1'i 2 shows the combined means and standard errors calcvlated from the two experiments.Inspection rf the data reveals significant effects on all parameters within the concentration range of50 to 1000 ug/niL. The effects on ATP and total protein levels were more pronounced than the effecton AM viability. Dose-response decreases in the response parameters with increased particleexposure were .een only in the 10- to 500-yg/mL concentration range. Exposure concentrations of 500and 1000 pg/mL appear to be equally toxic.

Statistical analysis of the data by one-way ANOVA indicated a main effect (p : 0.05) of exposureto the JCE0 particles for all end points. Subsequently, Williams' test, which requires a monotonicrelationship between exposure concentration and the measured response, was used to determine thedose at which an end point became significantly different (p5r 0.05) from control. The results shownin Table 2 demonstrate that the lowest effeccive dose for all parameters tested was 50 pg/mL ofJCE001 particles.

SUMMARY

The RAM test was used to determine the effects of the JCEOO1 particles on the percenta-e of AMviability, levels of ATP, and total protein. The presence of soluble cytotoxic components was alsoexamined. Incubation with the particles produced significant effects (p S 0.05) in AM viability,cellular ATP, and total protein levels. The lowest effective dose among all doses tested was 50 pg/mL.The presence of soluble cytotoxic components was established. The iature and rate of release ofsoluble toxic components from JCE001 particles were important contributing factors in themechanism by which th.e oarticles produced their toxic efficts.

PART;. THE EFFECTS OF JCE001 PARTICLES ON PULMONARY BACTERICIDAL ACTIVITY

OBJETIVE

The purpose of the study was to determine the effects of the JCE001 test particles on the ability of AMto kill inhaled [3 SSlKlebsiella oneumoniae.

MATERIALS AND METHODS

Test Particles

Atlantic brass, Richgold 1800, designated as JCE001 particles, was provided by the U.S. Army'Medical Research and Deve!opment Command and was stored at ambient temperature. According tothe data sheet that accomr'inied the test particles, the particles had a MMAD of 2.3 pm, with ageometric standard deviation of 1.61. The elemental analysis showed 65 to 74% copper, 26 to 35%zinc, and traces of lead, arsenic, aluminum, and various fatty acirs.,.

Because ofdifficul ties encountered in suspending the JCE001 particles for intratrachealU instillation, the particle suspersions were made by diluting a 10,000 pig/mL stock suspension. The

particles were first mixed ,nd wetted completely with IPA, then diluted in Medium 199 s, that the[PA was diluted to 1% of the total volume. The suspension was then sonicatedlhomogenized for 3 minusing a PT 10 sawtooth probe generator with a PTIO-35 Brinkmann homogenizer at power setting sixto break up the large agglomerates and produce a uniform suspension. Suspensicns (10, 50, 100, and200 jig) were made in 1% IPA ir FIBSS, which was also the diluent intratracheally instilled in thecontrol mice.

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TABLE 2. COMBINED EFFECTS OF 20-HR IN VITRO EXPOSURE AT 37C TO JCE001PARTICLES IN THE RAM TESTa

Exposure ATPConcentration Viability Total Protein fgx 106/pg

(pg/106AM/mL) (%) (pg/mL) fgX 108/mL, Protein

0 95.0 (1.6) 211.9(59.7) 11.18 (4.49) 5.16 (0.69)

10 85.6 (5.3) 210.4(63.3) 12.25 (6.18) 5.61 (1.27)

50 65.1 (10.9)* 97.3 (40.6)0 2.49 (1.36)*' 2.44 (0.36)*

250 28.4 (6.7)* 32.9 '.3.8)* 0.03 (0.01)* 0.11 (0.03)'

500 18.1 (4.1)1 32.3(13.2)0 0.15 (0.05)0 0.48 (0.04)0

1000 18.9 (4.4) 44.0 '(9.2)4 0.24 (0.08)* 0.57 ;3.30)0

a. Mean (± SE) are calculated from Experiments I and 2 in Table 1.* Significantly different from control (ps 0.05) determined by two-tailed Williams' test.

Thirty-day-old female CD-I mice weighing 19-21 g (Charles River Breeding Laboratories, Inc.,Kingston, NY) were quarantined for two weeks and we-e randomized into treatment groups at theinitiation of the study.

Intratracheal Instillation

Intratracheal instillation was performed as described by Aranyi et al.' Briefly, miceanesthetized with Metofane gas received 10-pL i, itatracheal injections of Medium 19911% IPA withor without the test compound. Suspensions of the particles were prepared by diluting each dose level.Aseptic procedures were used, and the suspensions were checked for sterility using standardmicrobiological procedures. The suspensions were used for intratracheal instillation within one weekof preparation to avoid agglomeration of the particles before injection.

Pulmonary Bactericidal Activity

Aerosols of [35SJK oneumoniae disseminated with a Retec X-70 disposable nebulizer were usedfor the bactericidal activity assay.' Radiolabeled K. oneumoniae were grown in a medium in whichthe sulfate requirement of the bacteria was provided by [35S]sodium sulfate. Pulmonary bactericidalactivity was determined in the lungs of the individual animals from both exposed and control groupsthat simultaneously inhaled aerosols of the viable radiolabeled bacteria. The ratio of the viablebacterial counts to the radioactive counts in each animal's lungs provided the rate at which bacteria'were destroyed 3 hr after infection. Thus,

R% Bactericidal Activity I R3- 100

where R3 is the ratio of bacterial-to-radioactive counts in the ,ings of individual mice at 3 hr, and K,,is an average determined from the same ratios in the lungs of four or five mice per group killedimmediately after inhaling the bacteria.

To achieve balanced exposure times to the particles from each dose level (Wt, 50, and100 pg/mouse), a rotation schedule was used whereby one mouse. from each dose group was injected insequence. Eighteen mice per dosage level received the particle suspension by intratracheal

j II

instillation. Twenty to 24 hr later, all mice were given simultaneously an inhalation challenge withan arosol of[35S]K, ojnumoniae. Four randomly selected mice per group wre killed immediatelyafter the challenge to determine the labeling ratio of the bacteria. The remaining 14 mice per groupwere killed 3 hr later and used to determine the pulmonary bactericidal activity.Statisticl Analysis

A three-factor mixed-model ANOVA was performed to examine the differential fixed effects oftest particles and dosage on bactericidal activity controlling for the randomeffect of replication.Bactericidal activity originally expressed as a percentage was natural log transformed to betterapproximate the assumed normality inherent in the use of this stacistical medaL

RESULTS

Twenty to 24 hr after intrstracheal instil!etion of the JCE001 particklt. the mice werechallenged with an aerosol of(3 sS]&. vneumoniae for the determination of bactericidal activity. Theresults are shown in Table 3. Although the bactericidal activity for all groups that received theJCE001 particles was reduced compared to the controls, there was no main effect oftreatment(p= 0.27). From these data, it appears that an increased sample size would be required to bring theobserved reversed dose-response to statistical significance.

TABLE 3. PULMONARY BACTERICIDALACTIVITY OF CD-I MICE TO INHALED

[35S]K. PNEUONIAE 20 HR AFTERINTRATRACHEAL INSTILLATION OF

JCE001 PARTICLES

JCE001 % Inhaled [35S]K. rniagmgmaalPg/Mouse Killed in 3 hr

0a 11.6 ± 2.9 (12)b

10 55.6 ± 8.4 (14)

s0 67.0 ± 5.3(13)

100 71.3 ± 4.6(12)

a. The controls received 1% IPA in Medium 199.b. Mean ± SE (number of mice).

SUMMARY

The effects of the JCE001 particles on the ability of AM to kill inhaled [SSIK. aneumoniae weremeasured. JCE001 particles (0, 10, 50, or 100 pg/mouse) were suspended in 1% IPA in Medium 199and intratracheally instilled in female CD-I mice 20 to 24 hr before aerosolchallenge with the[35S]K. oneumoniae. No statistical main effect of treatment was observed although the bactericidalactivity of all groups treated with the JCE00! particles was reduced compared to that of controls.

12

PART 3. THE EFFECTS OF JCE001 PARTICLES IN HAMSTER TRACHEAL RING ORGANCULTURE ASSAYS

OBJECTIVE

The objective of this study was to determine the toxic effects ofJCE001 particles ontrachoobronchial epithelium, as measured by cilia-beating frequency, morphological alterations, andbiochemical changes.

MATERIALS AND METHODS

The toxic elects of JCE001 particles on tracheal epithelium were assessed in hamster trachealring organ cultures.' Tracheal ring explants were allowed to stabilize for 24 hr prior to use.JCE001-containing medium was prepared at concentrations of 10, 50,250, and 1000 pg/mL. Becauseof the hydrophobic nature of the test material, the powder did not go into suspension. DMS0 wasthen used to prepare the suspensions. At 0 and I hr of exposure to JCE001-containing medium, thetracheaW specimens were examined for ciliary activity, cytotoxicity, and biochemical determinations,Specimens from each group were fixed and prepared for light microscopy and scanning electronmicroscopy.'

0

RESULTSCiliary Acti-vity and Cittopathology

The cilia-beating frequency of hamster tracheal rings exposed in vitro for I hr to variousconcentrations of JCE001 and observed immediately after is shown in Table 4. In initialexperiments, ring explants were exposed to JCE001 particles in L-15 culture medium. Exposure to aconcentration of JCEOO1 particles greater than 250 pz/mL resulted in a signifieant decrease in cilia-beating frequency. When explants were exposed to test material suspended -a DMSO-medium, theaverage cilia-beating frequency for each concentration was lower than particles in medium alone.. Asignificant reduction in ciliary activity was seen in explants exposed to a JCEOOI concentrationgreater than 50 pg/mL.

TABLE 4. MEAN CILIA-SEATING FREQUENCY IN HAMSTER TRACHEAL RINGORGAN CULTURES EXPOSED 1 HR TO JCE001 PARTICLES

Cilia Beating FrequencyConeent ation of (Beats/Min)

Test Particle L-15 Medium L-15 and DMSO Medium(•/mL) Meana SE Meane SE

Baseline 1253b 4 1239b 5Untreated control 1213b 5 1216b 6

10 1195bc 5 1189b.c 4

50 1166b,c 5 1156c.d 5250 1144d. 6 1125c.d 6

1000 1132d 12 1101e 23a. Mean data from 16 separate determinations.b,c,d. Values with the same superscript letter(s) within eacn column are not significantly

different (p : 0.65: Dunnett's test).

13

iU

Tracheal epithelium of explants exposed to medium or DMSO-mndium showed similarcytological alterations (Table 5). Significant alterations in the epithelium were seen with 250 and1000 pegmL ofJCE001. in areas of the epithelium not affected, beating cilia were observed. Focalareas of ciliostasis were seen in epithelium of explants exposed to a JCE001 concentration of1000 PWmL

TABLE 5. PERCENTAGE OF NORMAL EPITHELIUM IN HAMSTERTRACHEAL RING ORGAN CULTURES AFTER A I-HR IN VITRO

EXPOSURE TO JCE001 PARTICLES

Concentration of Percentage of Normal EpitheliumaTest Particle L-15 Medium L-15 and DMSO Medium

(•jg/mL) Meanb SE Meanb SEBaseline 9 1 954 2

Untreated control 93ei 1 9094 2

10 W9cA I "9d I

50 W5cd 2 6&,d 6

250 65. 6 W6. 61000 590 6 63a 6

a. Normal epithelium is defined as a smooth luminal surface with beating cilia.b. Mean data from eight separate determinations.c,d,e. Values with the same superscript letter(s) within each column are not

significantly different (p T. 0.05; x2 distribution test).

Histotathology (Licht Microm.'ow

Specimens exposed to the test mAterial in culture medium did not get the full impact of theparticles becauso they were hydrophobic and floated on the medium surface. Tracheal explantsexposed to all concentratiors exhibited no morphological changes as observed by light microscopy.The epithelium resembled that of control explants maintained in culture medium without testparticles.

The JCE001 particles were then prepared in spectrophotometr;.c-grade DMSO and added toculture medium. Histopethological examinations of untreated (DMSO-medium) control tracheasshowed ciliated, columnar epithelium, similar to that of normal hamster trachea immediately afterremoval from the animal., The epithelium #)f tracheal explants exposed to 10 and 50 .Ag/mL exhibitedoccasional vac'iolization. Exposure to greater than 50 pg/mL resulted in moderate pathologicalalterations. The epithelium showed some loss of ciliated cells, intracellular gaps, and vacuolization.Approximately 50% of the epithelium was not affected.

Scanninc Electron Microscopy

Untreated control trachea explants showed an epithelial surface consisting of ciliated andmicrovillous cells (Figures IA and 13). In JCEOO 1-treated specimens, particles were observed resting

* on the tips of the cilia'(Figures 2A and 2B). A second reaction was also observed involving the testparticles and the microvillous cells. Particles were occasionally observed attadhed to the microvilloussurface (Figure 3A). In other instances, particles detached from the microvillous surface leavingcrater-like structures (Figure 3B).

14

Figure IA. Cilated and microvillus cells of epithelium of control trachea explants, I I OX.

IA

~~ Figurvj 18. Ciliated anid rnicrovillus cells of epithelium of control trachea explants, 2200K.

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Figure 2A. Particles resting o tips of cilia in JCEOO1-treated trvicheal explants, 2200X.

Figure 2B. Particles resting o n tips of cilia in JCEOOI-treated tracheal explavits, 1IOiOX.

'Figure 3A. Particles attachedl to microvillus surface in JCEOOI-treatod tracheal explants, 2200X.

Figure 3B. Crater-Ilike structure in microvillous surface ofJICEOO I-treated tracheal explants. 55OOK.

Tritiated Thymidine Incorporation

Silver grains relating to incorporated tritiated thymidine were seen ever the nuclei of epithelialcalls in control (untreated) ring explants and in the epithelium of tracheal rings treated with 10, 50,and 250 pg•mL of JCE001 particles. This reaction indicates no inhibition of DNA synthesis.Inhibition of DNA synthesi, was observed in explants treated with 1000 pg/mL of JCEOOI particles.

Tritiated Uridine Incorporation

Silver grains over the nuclei and to a lesser extent over the cywplasms were seen in the controlexplante. Incorporation of tritiated uridine appeared in both ciliated cells and mucous cells, withheavier labeling indicating more RNA in the ciliated cells. Epithelia of tracheal rings treated withthe JCEO01 particlos were also labeled similar to those in controls.

Tritiated Leucine Incorporation

Silver grains corresponding to incorporated tritiated leucine were seen over the cytoplasm inboth the untreated control ring cultures and rings treated with all four concentrations of JCE001particles, indicating no inhibition of protein synthesis.Suecim'€ Qehvdroffnaes Activity (Histochemistry)

Untreated controls and JCE001-treated tracheas incubated in medium with the tetrazoliumsalt, succinate, edetic acid, and buffer showed succinin dehydrogenase activity as evidenced by a blueformazan stippling in the cytoplasm of epithelial cells.

SUMMARY

A single 1-hr in vitro exposure of hamster tracheal ring organ cultures to JCECOI particles wasconducted to determine the doss-responre on ciliated respiratory epithelium. JCE001 particlescaused a significant decrease in cilia-beating frequency and an increase in cytopathologicalalterations when tested at 250 and 1000 pg/mL. Morphological alterations in the tracheal epitheliumwere observed with light microscopy and scanning electron microscopy. An inhibitory effect ofJCE001 particles on RNA synthesis, protein synthesis, and succinic dehydrogenase activity was notdemonstrated. Only explant. treated with 1000 pg/mL of JCE001 particles showed an inhibition ofDNA synthesis.

PART 4. THE IN VIVO EFFECTS OF GRAPHITE PARTICLES ASBURY #850 AND DIXON KS-2ON PULMONARY BACTERICIDAL ACTIVITY

OBJECTIVE

The study was conducted to determine the effect of suspensions of two natural graphite samplesadministered by intratracheal instillation on the pulmonary bactericidal activity of mice to inhaled[35S]K. oneumoniae.

MATERIALS AND METHODSTe-st Purticles

Two natural graphite samples, designated Asbury #650 and Dixon KS-2, were provided by theU.S. Army Medical Research and Development Command and stored at ambient temperature. Eachparticulate sample first was mixed and wetted completely with [PA, then diluted in sterile saline sothat the fina. IPA concentration was 5% of the total volume. The suspensions were then dispersed for25 sec using a Model W186 sonicator (Sonifier Cell Disrupter Heat Systems - Ultrasonics, Inc.,

18

Plainview, NY) with a microtip at power settir 7' to break up the large agglomerates. The 5% IPAin sterile salne was also used as a vehicle control r intratracheal instillation in control mice.

Thirty-day-old female CD-I mice weighing 1 18 g (Charles River Breeding Laboratories, Inc.,Portage, MI) were quarantined for two weeks and andomized into treatment groups at the initiationof the study.Intratracheel Instillation

Intratracheal instillation was performed as 'bed by Aranyi et al.' Briefly, miceanesthetized with Metofane gas received intratra eal injections of 10 pL of 5% IPA/sterile salinewith or without the graphite particles. Suspensio of the particles were prepared by dilution foreach dose level. Aseptic procedures were used, an the suspensions were checked for sterility usingstandard microbiological procedures. The suspeni ions were used for intratracheal instillation withinone week of preparation to avoid agglomeration of the particles before injection.

Pulmonary Bactericidal Activity

Aerosols of [3ES]K. pneumoniae disseminate I with a Retec X-70 disposable nebulizer were usedfor the bactericidal activity assay.' Radiolabeled ,. oneumoniae were grown in a medium in whichthe sulfate requirement of the bacteria was provid.d by [35S]sodium sulfate. Pulmonary bactericidalactivity was determined in the lungs of the individ al animals from both exposed and control groupsthat simultaneously inhaled aerosols of the viable radiolabeied bacteria. The ratio of the viablebacterial counts to the radioactive counts in each nimal's lungs provided the rate at which bacteriawere destroyed 3 hr after infection. Thus,'

%BG clicidlA ity =(I ) 1oo

where R 3 is the ratio of bacterial-to-radioactive co te in the lungs of individual mice at 3 hr, and Kois an average determined from the same ratios in e lungs of mice killed immediately after inhalingthe bacteria.

Statisicl Analysis

A three-factor mixed-model ANOVA was per formed to examine the differential fixed effects ofgraphite articles and dosage on bactericidal activi y controlling for the random effect of replication.Bactericidal activity originally expressed as a per cntage was natural log transformed to betterapproximate the assumed normality inherent in Cis use of this statistical model."

Experimental Design s

The concentrations of the intratracheally injected test suspensions were selected based on theresults of preliminary LD 50 studies. Each graphite sample was tested at 25 and 250 pg/animal. Thetwo samples were tested simultaneously. Twelve mice per grou; were used with a total of sixtreatment groups in each replicate experiment: two for each of the two graphite samples, oneIPA/saline vehicle-control group, and one naive-cntrol group that received no intratrachealinstillation. Within 24 hr after intratracheal instillation of the graphite particles, mice from alltreatment groups were given a simultaneous inhslation challenge with an aerosol of[35SK. pneurnoniae. Two randomly selected nicl per group were killed immediately after thechallenge to determine the K, labeling ratio of th bacteria. The remaining 10 mice per group werekilled 3 hr later and used to determine the pulmor ary bactericidal activity. This experimental designwas repeated two more times to increase the sam~le size for statistical analysis.

RESULTS

A preliminary intratracheal instillation LD0, study was conducted using the Asbury #650 andDixon KS-? particles, each at 25, 100, 250, and 500 pg/mouse, along with the respective 2.5, 5, and

19

10% IPA/salin. vehicle controls. None of the mice died within 48 hr of the instillation. Mice thatreceived 500 pg of either particle appeared somewhat ruffled after 24 hr. The corresponding10% IPA/saline controls appeared normal. The dosage levels of 25 and 250 pg/mouse administered in5% IPA/saline were selected for both graphite samples in the final experimental design for the testsfor bactericidal activity.

The results of the bactericidal activity assays are summarized in Table 6. Statistical analysisfor comparing the two graphite samples was not significant (i.e., there was no main effect of testarticle or test article by dosage interaction). Analysis of the data for the Asbury #650 particlesshowed a main effect (p•;0.01) of treatment. Individual post hoc comparisons indicated the maineffect was caused by decreased bactericidal activity in the 250-pg treatment group relative to bothcontrol groups. The main effect of treatment for the groups that received the Dixon KS-2 particlesapproached significance (p!90.07). Once again, it was the 250-pg group whose bactericidal activitywas significantly decreased relativa to the naive controls (p:90.02 ) and the vehicle controls (p!90.03).Interpretation of the marginal main effect of treatment for the Dixon KS-2 groups is reasonable giventhe absence of a significant test particle by dosage interaction. Overall, the main effect of dosage inthe total sample (i.e., both test particles combined) was significant (p:90.025), and the two treatmentgroups of 25 and 250 pg also were significantly different (p ; 0.025).

TABLE 6. PULMONARY BACTERICIDAL ACTIVITY IN CD-i MICE TOINHALED [3 SSIK. PNEUMONIAE 24 HR AFTER INTRATRACHEALINSTILLATION OF ASBURY #650 AND DIXON KS-2 PARTICLES

Test Particle pgfMouse % Inhaled K. oneumoniae Killed in 3 hr

Naive controlsa 0 85.5 ± 1.3 (29)b

Vehicle controlsa 0 85.4 ± 1.5(28)

Asbury #650 25 84.3 ± 2.0(30)

Asbury #650 250 79.1 1 2.6 (30)**

Dixon KS-2 25 84.2 ± 1.8(29)

Dixon KS-2 250 81.0 ± 2.4(30)*

a. The naive controls received no intratracheal instillation.b. Mean ± SE (number of mice).c. The vehicle controls received 5% IPA in sterile saline.* p ! 0 .05 .** pvs 0.01.

SUMMARY

The effects of the Asbury #650 and Dixon KS-2 graphite particles on the pulmonary bactericidalactivity of mice to inhaled [35S]K. oneumoniae were measured. The particles (0, 25, or 250 ug/mouse)were suspended in 5% IPA/sterile saline and intratracheally instilled in female CD-I mice 24 hrbefore aerosol challenge with the (35S1K. pneumoniae. Both samples produced significantlydecreased bactericidal activities in the 250-pg treatment groups compared to both naive and vehiclecontrols. There was no significant difference between the effects of the intratracheally instilledAsbury #650 and Dixon KS-2 particles on the pulmonary bactericidal activity of mico.,

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¾ 4 .l,

L0ERATURE QUED

1. Hanks, J.H. and J.H. Wallace. 1958. A determination of cell viability. Proc. Soc. Exp. Biol.MmL 98:188-192.

2. Lowry, O.H., N.J. Rosebrough, AL. Farr, and R.J. Randall. 1951. Protein measurements withfolin phenol reagents. J. of Biol. Chem. 193:265-275.

3. Aranyi, C., J. Bradof, D.E. Gardner, and J.E. Lewtas. 1981. In vitro and in vivo evalvation ofpotential toxicity of industrial particles. In M.D. Waters, S.S. Sandhu, J.E. Lewtas, L. Claxton,and S. Nesnow, eds. Short-Term Bioassays in the Analysis of Complex EnvironmentalMixtures 1I. pp. 431-443, Plenum Press, New York, NY.

4. Williams, D.A. 1971. A test for differences between treatment means when several dose levelsare compared with a zero dose control. Biometrics 27(l):103-117.

5. Williams, D.A. 1972. The comparison ofseveral dose levels with a zero dose control.Siometrics 28(2):519-531.

6. Aranyi,C., J.L. Oraf, W. OShea, J.A. Graham, and F.J. Miller. 1983. The effects ofintratracheally admin - tered coarse mode particles on respiratory tract infection in mice. To&!Lrt. 19:63-72.

7. Aranyi, C., S.C. Vana, P.T. Thomas, J.N. Bradof, J.D. Fenters, J.A. Graham, and F.J. Miller.1983. Effects of subchronic exposure to a mi.-ture of O, S0 2, and (NH 4)2SO 4 on host defensesof mice. J. Tox. Environ. Health 12:55-71.

8. Winer, B.J. 1971. Multifactor experiments having repeated measures on the same elements.Chapter 7 in Statistical Princinles in Experimental Design. pp. 514-599, McGraw-Hill, NewYork, NY.

9. Schiff, L.J., M.M. Byrne, and W.T: Brown. 1978. Techniques tor determining ciliary activity inhamster tracheal organ culture. In V.J. Evans, V.T. Perry, and M.M. Vincent, eds. TssjjCulture Assoiation Manual. Vol. 4, pp. 871-874, Tissue Culture Association, Rockville, MD.

10. Schiff, L.J., M.M. Byrne, J.D. Fenters, J. A. Graham, and D.E. Gardner. 1979. Cytotoxiceffects of sulfuric acid mist, carbon particulates and their mixtures in hamster trachea)epithelium. Environ. Pes 19-339-354.

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