EPA-600/3-77-006
January 1977
Ecological Research Series
     PULMONARY CELL POPULATIONS IN  HAMSTERS
        MAINTAINED UNDER  EGYPTIAN  LABORATORY
                                           CONDITIONS
                              Environmental Monitoring and Support Laborator
                                     Office of Research and Development
                                    U.S. Environmental Protection Agency
                                          Las Vegas, Nevada 89114

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                 RESEARCH REPORTING SERIES

Research reports of the Office of Research and Development, U.S. Environmental
Protection  Agency, have  been grouped into five  series. These five broad
categories  were established to facilitate further development and application of
environmental technology. Elimination of traditional  grouping was consciously
planned to  foster technology transfer and  a maximum interface in related fields.
The five series are:

     1.    Environmental Health Effects Research
     2.    Environmental Protection Technology
     3.    Ecological Research
     4.    Environmental Monitoring
     5.    Socioeconomic Environmental Studies

This report has been assigned to the ECOLOGICAL RESEARCH series. This series
describes  research on the effects  of pollution on humans, plant and animal
species, and materials.  Problems are assessed for their long- and short-term
influences. Investigations include formation, transport, and pathway studies to
determine the fate of pollutants and their effects. This work provides the technical
basis for setting standards to minimize undesirable changes in living organisms
in the aquatic, terrestrial, and atmospheric environments.
This document is available to the public through the National Technical Informa-
tion Service, Springfield, Virginia 22161.

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                                             EPA-600/3-77-006
                                             January 1977
      PULMONARY CELL POPULATIONS IN HAMSTERS
  MAINTAINED UNDER EGYPTIAN LABORATORY CONDITIONS
                        by

 A. S. El-Sheikh, G. A. Abdel-Kader and S. 0. Amin
               Al-Azhar University
                   Cairo, Egypt
                       and
                  R. E. Stanley
Monitoring Systems Research and Development Division
 Environmental Monitoring and Support Laboratory
             Las Vegas, Nevada 89114
              Contract No. 03-546-1
                  R. E. Stanley
      x           Project Officer

Monitoring Systems Research and Development Division
 Environmental Monitoring and Support Laboratory
             Las Vegas, Nevada 89114
      U. S. ENVIRONMENTAL PROTECTION AGENCY
        OFFICE OF RESEARCH AND DEVELOPMENT
 ENVIRONMENTAL MONITORING AND SUPPORT LABORATORY
             LAS VEGAS, NEVADA 89114

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                                 DISCLAIMER
     This report has been reviewed by the Environmental Monitoring and
Support Laboratory-Las Vegas, U.S. Environmental Protection Agency, and
approved for publication.  Approval does not signify that the contents
necessarily reflect the views and policies of the U.S. Environmental
Protection Agency, nor does mention of trade names or commercial products
constitute endorsement or recommendation for use.

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                                INTRODUCTION

     It has been established that inhalation of various air pollutants in-
fluences the number and function of pulmonary alveolar macrophages (PAM)
(1-3).  The PAM constitute the overwhelming majority of the pulmonary cell
population and play an important role in the defense mechanism of the lung
against infection.

     This study was conducted to obtain baseline values for pulmonary cells
in golden hamsters (Mesoari-cetus auratus) bred and maintained under the
laboratory conditions of Al-Azhar University.  The data will be used for
comparison to that obtained from future studies concerned with the pulmonary
effects in hamsters following exposure to various inorganic air pollutants
indigenous to the Egyptian environment.

                                   SUMMARY

     In this study an improvised technique is presented for measuring pul-
monary cells obtained by lung lavage in golden hamsters (.Mesoaricetus
auratus),  The results of using this technique revealed a positive correla-
tion between the total count of pulmonary cells and the body weight of the
hamsters.  Cell differential counts showed that more than 99 percent of the
pulmonary cells were macrophages, with lymphocytes as the remainder.  The
findings are discussed and compared to those reported in the available
literature.

                                   RESULTS

     The absolute cell count (cells/mm3) and the calculated relative cell
count (cells/100 g body weight) obtained for each of the nine hamsters by
the technique described in this study are shown in Table 1.

     The average differential count for the nine hamsters was 99.34 percent
PAM and 0.66 percent leucocytes.

     The normal PAM harvested in the lung lavage varied in shape.  The cell
diameters ranged from 12 to 30 micrometers (vim), and the diameters of the
nuclei ranged from 9.6 to 13.2 urn.  As shown in Figure 1, the nuclei are
deeply stained and eccentrically located.  The cytoplasm is basophilic and
shows ingested dust particles in several cells.  In Figure 2, it is shown
that the cell contours vary from oval to irregular, and occasional cytoplasmic
processes are also seen.

                            MATERIALS AND METHODS

     Nine male golden hamsters selected from breeding stock obtained from
the Naval American Medical Research Unit at Abbassia, Egypt, and from the

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TABLE 1.  ABSOLUTE AND RELATIVE TOTAL COUNTS OF PULMONARY CELLS IN NINE GOLDEN
                                  HAMSTERS
Hamster
Number
1
2
3
4
5
6
7
8
9

Number of Replicate
Counts from Stock
Suspension
11
9
6
6
5
4
3
7
4
Average
Body
Weight
(g)
60
80
77
75
170
170
170
170
170
127
Mean Absolute
Count
(cells/mm3)
874
1043
1140
1068
2410
2000
2800
2657
2575
1841
Calculated Relative
Count (cells/100 g
body weight)
1457
1304
1481
1424
1418
1176
1647
1563
1515
1443
 Figure 1.  Cell nuclei deeply stained  Figure 2.  Oval cells with occasional
 and eccentrically positioned. 280X.    visible cytoplasmic processes.  320X.
                                        2

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Ministry of Health Laboratory for Serum Production at Helwan, Egypt, were
used in the study.  The hamsters were housed individually in galvanized wire
cages under standard conditions of room temperature (221C) and relative
humidity (505%).  The ration fed to the animals consisted of a mixture of
fresh and commercially processed food containing approximately 20 percent
protein, with water available ad libitum.   Body weights ranged from 60 to
170 g.

     The technique used for harvesting the alveolar pulmonary cells is a
modification of those used by Myrvik et al. (4) and by Coffin et al. (5).

     The hamsters were anesthetized by intraperitoneal injection of sodium
pentobarbital (6.5 mg/100 g body weight).   The trachea was exposed and in-
cised slightly posterior to the larynx.  A polyethylene cannula was inserted
into the trachea and maintained in place with an annular ligature.  Necessary
precautions were taken to avoid any contamination by blood within the trachea
or lungs.  Three to four ml of normal saline (0.9 percent NaCl maintained
at 37C) with a few drops of gentian violet added to stain the nuclei of
the cells to make them visible, were injected through the cannula into the
lungs and allowed to remain for 15 minutes.  The lavage fluid was then with-
drawn from the lungs, using a syringe, and ejected into a graduated conical-
shaped centrifuge tube which was positioned in a crushed-ice bath.  The lung
washing was repeated five more times in a similar manner, except that the
lavage fluid was withdrawn from the lung immediately after each instillation.

     The lavage fluid collected in the centrifuge tube from the six washings
was centrifuged at 1500 rpm for 20 minutes.  Following the centrifuging, the
supernatant fluid was removed using a capillary pipette.  The remaining cell
pellet was then resuspended in 3 ml of normal saline and the mixture agitated
by inverting the centrifuge tube until the cell pellet was no longer visible.
The cell suspension was recentrifuged for 15 minutes, and the supernatant
fluid was again removed with a capillary pipette.  After this washing, the
cell pellet was resuspended in 1 ml of normal saline using a gentle current
of air as the source of agitation to effect the suspension.  This suspension
was used as the stock suspension for the total and differential counts and
for the source of macrophages studied in the purified form.

     The total cell count was accomplished using exactly the same procedure
as that used for counting white blood cells.  The cell suspension (stock
suspension) was aspirated into a hemocytometer pipette and diluted  (10 to 1)
with normal saline containing a few drops of gentian violet.  One drop was
then placed on the counting chamber, covered with a cover slip, and the cells
counted.  After several replicate counts, the mean count (cells/mm3) was used
to calculate the total number of cells in the stock suspension.  This abso-
lute cell count was then used to calculate the relative count (cells/100 g
body weight).

     The cell differential count was made from the stock suspension, also.
One drop of the stock suspension was spread over a clean glass slide, air
dried, fixed with formalin vapor, and stained with Giemsa stain.  The dif-
ferent types of cells (PAM, lymphocytes and neutrophils) were counted in
each field and tabulated as to the relative percentage observed.

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                                 DISCUSSION

     This study shows that the absolute pulmonary cell count of the stock
suspension seems to vary directly with the body weight, and that the rela-
tive count calculated according to body weight was more or less constant.
This finding indicates a positive correlation between total pulmonary cells,
obtained by lung lavage, and body weight.  This correlation is logical when
considering the relationship between alveolar surface area and body mass.

     As can be discerned, these apparent conclusions were drawn without
benefit of any formal statistical analysis and appear obvious.  Although
the concept of the absolute cell count as a function of body weight is
clearly established, the data were analyzed to confirm these obvious con-
clusions using a modified analysis-of-variance program considering all
hamsters combined and all possible comparisons of all pair combinations.
Testing the hypothesis of no difference among hamsters when the cell count
is adjusted for body weight, the probability of this hypothesis being true
changes from 3xlO~21f percent to 4.4 percent, a 1.5xl02^-fold improvement in
the consistency of the data due to the weight adjustment.  Therefore, the
improvement is so gross and obvious that statistical analysis was, in this
case, superfluous.  However, the authors are the first to admit that the
number of animals used was less than desirable.  The almost total absence of
body weights in the 80-to 170-g range does not permit one to definitely
establish the functional relationship indicated.

     Myrvik et at. (4) using their own technique to obtain PAM in a high
state of purity, reported that in the rabbit, the total yield from normal
lungs usually varied between 0.1 and 0.2 ml of packed cells (8 to 16 million
cells).  Coffin e~t at.  (5), however, reported the number of cells obtained
from unexposed rabbits' lungs as high as 43.5 million with a standard error
of 4.7 million.  The rabbits used by these two groups of investigators were
more or less the same weight.  Nevertheless, the obvious discrepancy in
their findings could be attributed to the different techniques employed by
the two groups of workers, strain differences in the animals, and/or dif-
ferences in individual laboratory conditions.

     This work, however, presents a method of relating cell count to body
weight, which tends to greatly minimize the differences reported when based
on absolute count alone.

     PAM constituted more than 99 percent of the total pulmonary cells re-
covered from the lung by lavage.  Less than 1 percent of the recovered pul-
monary cells were lymphocytes.  Polymorphs and eosinophils were difficult to
find in any of the smears.

     By comparison, Gardner et at. (6) found that in the unexposed rabbit,
98 percent of the pulmonary cells were mononuclear macrophages.  Small
lymphocytes constituted 1 percent of the total observed, while occasional
polymorphonuclear leucocytes and eosinophils comprised the remaining 1 per-
cent.  Myrvik et at. (4) reported that in the rabbit, also, the cell sus-
pension contained less than 1 percent polymorphonuclear cells, with the
alveolar macrophages constituting essentially all of the cells observed.
Myrvik et at. (4) further stated that lymphocytes were seldom seen.

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     In smears stained with hematoxylin and eosin, Myrvik &t at.  (4) described
the general morphology of alveolar macrophages obtained from rabbits' lungs as
resembling plasma cells rather than blood monocytes.  They further reported
variation in size of the macrophages, although the cells possessed a strikingly
uniform appearance.  In this study, the Giemsa-stained material showed the PAM
to vary both in size and uniformity.  In fact, variations in shape and contour
among the cells were even more pronounced than the variation in size, and
protoplasmic processes were easily seen in some cells.  None of the cells exam-
ined were binucleated or trinucleated as was reported by Myrvik et al. (4).
                                 REFERENCES

1.  Gardner, D. E., T. R. Lewis, S. M. Alpert, and D. L. Coffin.  "The Role of
          Tolerance in Pulmonary Defense Mechanisms."  Arch Environ Health
          25.  1972.

2.  Waters, M. D., D. E. Gardner, and D. L. Coffin.  "Cytotoxic Effects of
          Vanadium on Rabbit Alveolar Macrophages In Vitro."  Toxicol Appl
          Pharmaeol 28.  1974.

3.  Coffin, D. L., and D. E. Gardner.  "Interaction of Biological Agents and
          Chemical Air Pollutants."  Ann Oooup Hyg 15.  1972.

4.  Myrvik, Q. N., E. S. Leake, and B. Fariss.  "Studies on Pulmonary Alveolar
          Macrophages from the Normal Rabbit:  A Technique to Procure Them
          in A High State of Purity."  J Irmunol 86.  1961.

5.  Coffin, D. L., D. E. Gardner, R. S. Holzman, and F. J. Wolock.  "Influence
          of Ozone on Pulmonary Cells."  Arch Environ Health 16.  1968.

6.  Gardner, D. E., R. S. Holzman, and D. L. Coffin.  "Effects of Nitrogen Di-
          oxide on Pulmonary Cell Population."  J Bacterial 98.  1969.

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                                   TECHNICAL REPORT DATA
                            (Please read Instructions on the reverse before completing)
1. REPORT NO.
 EPA-600/5-77-OQ6
                                                           3. RECIPIENT'S ACCESSION-NO.
4. TITLE AND SUBTITLE

 PULMONARY CELL POPULATIONS IN HAMSTERS MAINTAINED
 UNDER EGYPTIAN LABORATORY CONDITIONS
                                                           5. REPORT DATE
                                                             January 1977
                                                           6. PERFORMING ORGANIZATION CODE
7. AUTHOR(S)
 A.S El-Sheikh, G.A.  Abdel-Kader and S.O. Amin (block
 9);  and R.E. Stanley  (block 12)
                                                           8. PERFORMING ORGANIZATION REPORT NO.
9. PERFORMING ORGANIZATION NAME AND ADDRESS
                                                            10. PROGRAM ELEMENT NO.
  Al-Azhar University
  Cairo, Egypt
                                                           11. CONTRACT/GRANT NO.

                                                             Contract No. 03-546-1
 12. SPONSORING AGENCY NAME AND ADDRESS
  Environmental Monitoring and Support Laboratory
  Office of Research and Development
  U.S.  Environmental Protection Agency
  Las Vegas, Nevada  89114
                                                           13. TYPE OF REPORT AND PERIOD COVERED
                                                           Progress-Calendar Year 1974
                                                           14. SPONSORING AGENCY CODE
                                                            EPA - Office of
                                                            International Affairs
 15. SUPPLEMENTARY NOTES
  This study was  supported by the Special  Foreign Currency Program, P.L.  480.
 16. ABSTRACT

  This study was  conducted to obtain baseline values for pulmonary cells  in golden
  hamsters (Mesocricetus auratus) bred and maintained under the laboratory  conditions
  of Al-Azhar University in Egypt.  An improvised technique is presented  for measuring
  pulmonary cells obtained by lung lavage in golden hamsters.  The results  of  using
  this technique  revealed a positive correlation between the total count  of pulmonary
  cells and the body weight of the hamsters.   Cell differential counts  showed  that
  more than 99 percent of the pulmonary  cells were macrophages, with lymphocytes as
  the remainder.   The findings are discussed and compared to those reported in the
  available literature.
17.
                                KEY WORDS AND DOCUMENT ANALYSIS
                  DESCRIPTORS
                                              b.lDENTlFIERS/OPEN ENDED TERMS  C. COSATI Field/Group
     lung
     cell morphology
     phagocytes
     laboratory animals
                                                pulmonary alveolar
                                                   macrophages
                                                lung  lavage
                                                hamster
                            06 C, P
                            14 B
                                                                         21. MO. OF PAGES
                                                                               8
18. DISTRIBUTION STATEMENT

    RELEASE T,0 PUBLIC
19. SECURITY CLASS (ThisReport)
  UNCLASSIFIED
                                               20. SECURITY CLASS (Thisp*ge)
                                                 UNCLASSIFIED
                                                                         22. PRICE
EPA Form 2220-1 (9-73)

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