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47.
81
OR Na-P. 81
DILL
Radiation Research
JUAL 10 1864
MASTE
Thirty-Day Mortality of Acutely X-Irradiatea "Germfree" ICR Mice*
8. I. Walburg, Jr., E. J. Mynatt, and D. M. Robie
Biology Division, Oak Ridge National Laboratory, Oak Ridge, Temessee

This paper was submitted for publication
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* Research sponsored by the U.S. Atomdc Energy Conmi 88ion under contract
with the Union Carbide Corporation.
Send proor to: Dr. H. E. Walburg, Jr.
Biology Division
Oak Ridge National Laboratory
P. 0. Box Y
Oak Ridge, Tennessee 37831
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INTRODUCTION
Miller (1) has described and offered an explanation of the pathogenesis
of the endogenous bacteremias caused by enteric microorganisms following
exposure to acute lethal radiation, suggesting that the enteric flora plays
a significant role in radiation sickne86. Antibiotics capable of suppressing
some elements of the bacterial flora have been shown to reduce the mortality
in irradiated mice (2). Preliminary studies showed both increased survival
and increased survival time in irradiated germfree rats and chickens as
compared with conventional controls (3, 4) and irradiated germfree mce showed
increased survival and survival time when compared with mice contaminated
with an E. coli three weeks prior to irradiation (5). However, X-irradiated
germfree and conventional Swiss-Webster mice did not show a marked difference
in thirty-day mortality, and the survival time of the germfree mice in the
LD-200 range was typical of that seen in conventionally reared mice (6).
demonstrate a marked increase in their LDCo/2o and in their survival time.
MATERIALS AND METHODS
Germfree and conventional noninbred ICR mice were reared and maintained
in the Biology Division of the Oak Ridge National Laboratory. Their ancestors
were germfree ICR breeders supplied by the Lobund Laboratory, University of
Notre Dame. The germfree mice were housed in plastic-film isolators (7)
sterilized with ethylene oxide and were maintained by a modification of the
routine procedures developed at the Lobund Laboratory. Surveillance for
contand nation consisted of periodic aerobic and anaerobic culture of feces
and tissue for bacteria, mycoplasma, fungi, and yeast, examination of
.(
intestinal contents and the skin for parasites, and scrological examination
for t..e following viruses:
Polyoma, mouse hepatitis, Sendai Theiler's,
pneumonia virus mouse, "K", mouse adenovirus, Rcovirus 3. The conventional
mice were derived from ancestors which had been removed from an j.solator
and placed in a breeder room with conventional mice at least one and one-half
years prior to this experiment. Standard husbandry techniques were followed,
except that the mice were fed the same sterilized diet as the germfree mice
(Purina Lab Chow, 50100). Microbiological studies were not carried out on
these mice.
Ten- to twelve-week-old germfree mice were irradiated in a sterile
isolator while confined on a turntable under the X-ray beam. The conventional
mice were irradiated under identical conditions but were then removed from
the isolator and returned to conventional surroundings. The GE Maxitron X-ray
machine was operated at 300 kvp and 20 ma, with additional filtration of 1/4 mm
Cu and 1 mm Al (HVL = 0.98 mm Cu), at a dose rate of about 100 R/min. Following
removal of the mce from the irradiation isolator, an unsterlle Victoreen
condensor dosimeter was introduced for determination of the dose rate. Cages
were observed for dead animals twice daily. .
RESULTS
The effect of acute X-Irradiation on survival in the LD-to-ID 200 range
is seen in Table 1. Data for males and females are not significantly different T-1
and are pooled for analysis. The LD 50/30 values as determined by probit
analysis are 739.2R for conventional mice and 899.2R for germfree mice, a
difference of 160R (see Table 2). The slopes of the dose-mortality curves T-2
are significantly different, 17.37 x 10°3 for the conventional mice and
P-1.
9.37 x 10-3 for the germfree mice (F48. 1). The mean survival time of mice
dying during the first month after irradiation with doses from LD, o to LD, 100
ranged from 11.36 to 12.10 days in conventional mice and from 14.47 to 19.20
days in germfree mice. The standard error of the mean survival time is
consistent.
Wen
r in the a
ventional mice.
A
time
distribution of deaths of the irradiated germfree and conventional mice within
dose ranges yielding comparable mortality demonstrates earlier and less
scattered. deaths in the conventional mice (Fig. 2).
F-2
DISCUSSION
The LD50/30 for Irradiated conventional ICR mice reported here is typical
of the LD50/30 reported for conventional mice irradiated in other laboratories
(500-770 R) (6, 8, 9, 10, 11). The ID 50/30 of 900 R for the germfree ICR
mice is considerably above these values and suggests an effect of the germfree
state on survival after irradiation. The significance of the microbial flora
18 further underscored by the 60-70% increase in percentage of the germfree
mice surviving, as compared with their conventional counterparts, at dose
levels of 800-850 R X-radiation.
Experiments in other laboratories have led to the various conclusions
X-irradiation is independent of mouse strain (11) or varies with strain
(8, 9). The difference in slope of the dose-survival curve for germfree
and conventional ICR mice may reflect a role of the microbial flora and
suggests that the variations seen in the dose-survival curves of conventional
mice are at least partly attributable to differences in microbial flora.
The mean survival time of the conventional ICR mice in these experiments
is typical for conventional mice receiving LD.10 to LD.,10. doses of X-radiation,
1.e., 8-16 days (€, 12, 13). The range of the mean survival tive of the
Irradiated germfree ICR mice is significantly higher than that of the
conventional controls and is about 2 days higher than that reported for
conventional mice from other laboratories.
These alterations of the survival patterns of germfree mice exposed
to LD, to LD,
doses of X-radiation support the conclusion that bacterial
flora play a significant role in the pathogenesis of radiation sickness,
as suggested by the development of postirradiation bacteremias caused by
enteric microorganisms, by the evidence that bacterial type effects the
duration of life after the onset of these bacteremias, and by the protective
effect of antibiotics administered to lethally irradiated mice (1).
SUMMARY
Germfree and conventionally reared adult ICR mice were exposed to
600-1000 R whole body X-radiation. The LD/30 was 739.2 R for conventional
mice and 899.2 por germfree mice. Both the LD50/30 and the slope of the
mortality curve of the germfree mice were significantly different from those
of the conventional mice.
The elevated LD50/30 and extended mean survival
time seen in germfree mdce support the conclusion that the bacterial flora
play a significant role in radiation disease.
ACKNOWLEDGEMENT
The assistance of M. A. Kastenbaw in the statistical analysis of this
data and of E. tetter in the serological survey for viruses 18 gratefuly
acknowledged.
We wish to express our sincere appreciation to the Lobund Laboratory,
University of Notre Dame, and especially P. C. Trexler, R. Hakes and J. Wiley
for providing both the nucleus breeding stock for our germfree colony and
the technical knowledge which enabled us to maintain it.
..
REFERENCES
1. C. P. Miller, The effect of irradiation on natural resistance to
infection. Ann. N. Y. Acad. Sci., 66(2), 280-291 (1956).
2. C. P. Miller, C. W. Hammond, M. Tompkins, and G. Shorter, The treatment
of post-irradiation infection with antibiotics: An experimental study
on mice: J. Lab. clin. Med. 39, 462-479 (1952).
3. J. A. Reyniers, P. C. Trexler, W. Scruggs, M. Wagner, and H. A. Gordon,
Obsetvations on germfree and conventional albino rats after total body
X-radiation. Radiation Research 5, 591 (1956) abstract.
4. M. M. McLaughlin, M. P. Dacquisto, D. P. Jacobus, M. Forbes, and D. E.
Parks, The effect of the germ-free state or survival by the ten-day-old
chick exposed to X-ray. Radiation Research 2, 147 (1958) abstract.
5. M. M. McLaughlin, M. P. Dacquisto, D. P. Jacobus, R. E. Horowitz, and
S. M. Levenson, The effect of the germ free state on survival of mice
exposed to X-ray. Radiation Research 14: 484 (1961) abstract.
6. H. Frålen, K. Bö Lining and C. Rönnböck, The effect of X-irradiation on
various mouse strains due to their genetic background. l. Lethality
after acute irradiation. Radiation Research 14, 381-393 (1961).
7. P. C. Trexler and L. I. Reynolds, Flexible fllm apparatus for the rearing
and use of germfree animals. Appl. Microbiol. 5, 406-412 (1957).
8. D. Grahn and K. F. Hamilton. Genetic variation in the acute lethal
response of four inbred mouse strains to whole body X-irradiation.
Genetics 42, 189-198 (1957).
9. D. Grahn, Acute radiation response of mi.ce from a cross between
radiosensitive and radioresistant strains. Genetics 43, 835-843 (1958).
10. W. H. Chapmaja, The weight and mortality response of male and female mice
in the lethal X-ray dose range. Radiation Research 3, 502-51 (1955).
1. H. I. Kohn and R. F. Kallman, The influence of strain on acute X-ray
lethality in the mouse. 1. LDC, and death rate studies. Radiation
Research 5, 309-317 (1956a).
12. 8. I. Kohn and R. F. Kallman, Relative biological efficiency of 1000-KUP
and L50-KUCP X-rays. III. Determinations based on the LD50/28 and the
kalling time of the mouse. Radiation Research 5, 693-699 (1956b).
13. E. P. Cronkite, The diagnosis, prognosis and treatment of radiation
injuries produced by atomic bombs. Radiology 56, 661-669 (1951).
.
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TABLE I
Thirty-Day Mortality and Mean Survival Time of X-Irradiated ICR Mice

Germfree Mice
NO. Dead
Mean Survival
Conventional Mice
No. Dead
16
No. Irradiated Mortality*
No. Dead
Dose (R)
Sex
No. Irradiated
Mortality*
Time * S.E. X
Sex
Mean Survival.
Time t S.E.X
sue
1000
1000
5/5
5/7
15.80 $ 1.02
,
f
950
65.4
14.47 $ 0.91
3/10
3/6
900
900
55.6
18.12 + 1.23
$
7/7
100.0
200.0
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6
850
30.4
17.00 $ 1.07
a
100.0
21.53 + 0.66
10
10/10
7/7
6/6
; 9/9
to ot :
TABLE I (continued)
Germfree Mice
No. Dead
No. Irradiated Mortality#
Conventional Mice
No. Dead
No. Irradiated Mortality#
Dose (R)
Mean Survival
Time S.E. X
Bex
Sex
Mean Survival
Time : S.E. X
16.1
19.20 $ 1.21
80.5
12.70 1 0.39
1/10
3/7
1/7
017
$
8/10
6/8
476
6/7
9/10
0/8
9/10
750
54.3
21.63 + 0.97
12
3/8
3/7
4/10
5/9
700
6
0/8
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1/7
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11.36 0.64
0/7
7/10
1
0/8
650
0/6
0.0
0/10
* oland
and different experiments pooled at each dose.
12
TABLE II
Table of Numerical Results
Estimates
Germtree
Conventional
LD50/30
Lower 95% C.I.
Upper 95% C.I. :
slope
8.B. of slope
899.2
876.4
927.4
9.37 x 10-3
1.62 x 10-3
739.2
725.0
752.6
17.37 x 10-3
2.21 x 1003
30-day MORTALITY (%, PROBITS)
-13-
mortality
Jeg I Thinke in Sawid donfule and
converurnal ICR nice in sifatin to..ASSIFIED
* xray Quee levil.Cows 785'm 17.22982 19.69253
CONVENTIONAL
---
30-0 MORTALITY (%, PROBITS)

GERM FRSE
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600
700
800
DOSE (r)
900
1000
i
Kos 10 X 10 TO THE CM. 358.14
KoE keur TCL, O EGBER CO. "
Fig. 2.
Tinie dretriebutess of deaths of ICR
miel exposed to single exposures gx-radiators in the LDG-6-(Divo Range
ویه 3

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This paper was submitted for publication
in the open literature at least 6 months
prior to the issuance date of this Micro-
card. Since the U.S.A.E.C. has no evi-
dence that it has been published, the pa-
per is being distributed in Microcard
form as a preprint.
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DATE FILMED
16/28/65






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