Vol. 67, No. 3, 1975
BIOCHEMICAL
RADIOPROTECTION
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
OF BONE MARROW STEM CELLS BY SUPEROXIDE DISMUTASE
A. Petkau, K. Kelly', and B. E. Meeker
W. S. Chelack,
S. D. Pleskach,
C. Barefoot?,
Medical Biophysics Branch, Whiteshell Nuclear Research Establishment, Atomic Energy of Canada Limited, Pinawa, Manitoba, Canada ROE 1LO tImmunology Department, University of Manitoba, Winnipeg, Canada Received
October
16,1975
SUMMARY In mouse bone marrow cells the radioactivity from intravenously injected 125 I - superoxide dismutase reached its maximum level 1 h later. The 1 h interval was used while examining the effect of the unlabelled enzyme on the proliferative capacity of X-irradiated hematopoietic stem cells, as measured by When administered to donor mice their ability to form colonies in host spleens. in the amount of 35 ngfg body weight, the enzyme protected the cells, exposed to 350 rads, by a factor of 2.16 50.18. At doses of 15, 70, and 100 pg/g, this factor was reduced to 1.44 + 0.18, 1.15 t 0.15, and 0.7 + O.l,respectively. The value of 2.16 at 35 pg/g was increased to 2.5 by giving an additional therapeutic dose of the same size 1 h after the X-irradiation. INTRODUCTION
Recently,
it
was shown
superoxide
dismutase
protected
of ionizing
radiation
(1).
dose range
where
This
depression
of hematopoiesis
survival.
effect
of the enzyme on the blood
protect colony
of its
association
suggested
with
the proliferative assay
technique
white
protection
animal
terms
It
Swiss
that
the
present forming
bone marrow
capacity
of these
an intravenous mice
dose of bovine
from the
was elicited is study organ
the
effects
within
a radiation
principal
in which in mice
stem cells cells,
lethal
defect
governing
the radioprotective is
examined
and in its as measured
ability
both
in to
by the spleen
(2).
MATERIALS AND METHODS C3H/HeB inbred male mice weighing 16-22 grams each, were obtained from RioBreeding Laboratories of Canada Ltd., Ottawa, and from Canadian Breeding Farms and Laboratory Ltd., St. Constant, Quebec. Each shipment of mice, upon arrival, was divided randomly into donor and host groups and given food and water ad libitum, The host mice were housed in groups of 3 per cage. Lyophilized superoxide dismutase, obtained from Truett Laboratories, Dallas, Texas was dissolved in sterile normal saline (0.9%) for intravenous administration. In the solution, the enzyme concentration was 0.60, 1.4, 2.8, or 4.0 mgfml depending on whether a 20 gram donor mouse was to receive 15, 35, 70, or 100 ng of enzyme per gram body weight, respectively, in an injected (via tail vein) volume of 0.5 ml. Enzyme-treated donor mice of lesser or greater weight received proportionately smaller and larger volumes, respectively, Control donor mice were injected either 1 h before or 1167
Vol. 67, No. 3, 1975
1 h after
BIOCHEMICAL
X-irradiation
or at both
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
times
Fn tandem.
Donor mice were exposed to 350 rads, at a rate of 100 with 250 KV, 15 mA X-rays filtered by 0.5 mm Cu and 1 mm Al. received no prior treatment and were supralethally irradiated with X-rays of identical quality during the evening preceding which they were intravenously injected with donor bone marrow
rad/min, Host mice to 800 rads the day in stem cells.
One hour after completion of the irradiation or post-exposure treatment, the donor mice were sacrificed and the femora removed. The bone marrow stem cells from each femur were eluted as described previously (2) with 0.5 ml of Hank's balanced salts (HBS) solution. In any particular experiment, samples from individual mice in each treatment category were pooled and stored on ice prior to use. The number of nucleated cells in the pooled samples was counted in a hemocytoneter. Each sample was divided into two parts with adjustment in the cell number in one part to 18 x 106 and in the other part to 4.5 x 106 cells/ml, by dilution with HBS solution. The diluted cell suspensions were injected by tail vein into the host mice, with each mouse receiving 0.5 ml. Each dilution was injected into a minimum of 27 mice. The host mice were observed for 10 days during which some of them died. The surviving fraction was generally larger in groups that had received donor stem cells treated with superoxide dismutase rather than 0.9% saline. The survivors were sacrificed, the spleens removed and fixed in Bouin's fixative. The number of colonies on the surface of each spleen was counted with the aid of a low power (20X) stereomicroscope. In instances where the colonies were too numerous and confluent, the spleen weight was taken as an indicator of stem cell proliferative activity. During the course of this study the chloramine-T technique (3) was used to label the tyrosine residues of bovine superoxide dismutase with =51. The chemicals required in the preparation were bovine superoxide dismutase ((SOD), Truett Laboratories) and reagent grade chloramine-T, sodium metabisulfite, potassium iodide, and 0.05 M phosphate buffer, pH 7.5. Sodium (lz51) iodide was obtained from New England Nuclear Canada, Dorval, Quebec. It was supplied in aqueous solution, p Ii 8 - 10, and specified as thiosulfate-free as well as carrier-free, with an isotopic purity > 99%. The reagents were freshly prepared in the 0.05 M phosphate buffer to the following concentrations per ml: SOD 8 mg, chloramine-T 4 mg, Na2S205 2.4 mg, and KI 10 mg. To the reaction vial, containing 2 mCi of Na 1251/0.0045 ml, were added 0.025 ml each of 0.05 M phosphate buffer (pH 7.5), SOD, and chloramine-T solutions with brief mixing after each addition. Following the addition of chloramine-T, the reaction was allowed to proceed for 5 min before 0.1 ml of Na2S205 and 0.2 ml of KI were added. The labelled enzyme was separated from free 1251 on a Bio-gel P-60 column, equilibrated beforehand in 0.05 M phosphate buffer and presaturated with bovine serum albumin (25 mg/0.5 ml) to eliminate absorption of the labelled enzyme. the latter was collected in 0.5 ml fractions which were The labelled enzyme was pooled as indicated ~,"~?de:~:iY~~I activity. (Fig. 1) and further purified by exhaustive dialysis against physiological The labelled enzyme had an activity of 5 x 10' saline (0.15 M, pH 7.4). cpm/ml, equivalent to 3.4 x lo6 cpm/l.lg SOD. It was administered to mice in uptake studies in which the organ distribution and cellular uptake of the labelled enzyme could be followed. The small quantities (0.1 - 0.3 The mice were ml/mouse) of the 1251-SOD were injected intravenously. sacrificed at various times thereafter to collect serum and bone marrow stem cells for the purpose of counting their 1251 activity. RESULTS
The elution
is
in Fig.
given
profile
1 which
of the 125 I-labelling
shows
that
more than
1168
reaction 50% of the
mixture radionuclide
BIOCHEMICAL
Vol. 67, No. 3, 1975
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
IO-
I
2-
IO
also
on Rio-gel I-labelled
to the superoxide
dismutase
separated
f/15-40 used
were
from pooled
to obtain
mouse serum
the
and,
the
after
in-vivo
the amount
paralled
level
with
the eluted
eluted
its
drop
Removal
of the
remaining
with
in the
cells
former
70
00
data
W phosphate dismutase
labelled
the 125
of
physiological
of Fig.
2.
It
enzyme decreased
tubes saline,
shows
after
of eluted
The amount
to increase
that
half
suspensions
contained
by differential
the nucleated
cells.
during
both
the the
erythrocytes lysis
left
When the
1169
in an hour
bone marrow
directly
within
declined associated hour
first
first
four
41% of the were
125
lysed
in with
and was hours.
and nucleated
latter
buffer (1251-SOD)
enzyme was
I-SOD within
against
In suspensions
9c
to a maximum at 1 h and thereafter
appeared
total
bone marrow
and that
in the serum.
also
60
P-60 column, 0.05 bovine superoxide
dialysis
of labelled in 6 h.
40 50 NUMBER
Fractions
.
uptake
increased
cells
10 - 18% of the
125I
free
and was Q 90% cleared cells,
30 TUBE
.
Figure 1. E1utyy5yf;lf25 pH 7.5, of free
was bound
20
The cells.
I activity and centri-
BIOCHEMICAL
Vol. 67, No. 3, 1975
AND BIOPHYSICAL
Figure 2. Variation with time of 1251-labelled SOD) in serum (n ), LHS ordinate), eluted dinate), and bone marrow stem cells (X-X), present standard deviations.
fuged
(1 h at 1000,000
x g),
and the particulate
nuclei,
accounted
etc.)
radioactivity strate from
of the
that gel
the
is
The increase superoxide
dismutase
was also
accompanied
of the given
tissue
the
in Table
1,
with
to a factor
treated
with
one-half
times,
results
amount
before
that
of exogenous stem
cells
effect The data,
radioprotective
respectively,
30-70%
extent.
radioprotective
of the cells.
that
suggests
bone marrow
and one hour
1170
It
and low
to demon-
indicated
to a small
this
volume
SOD,preliminary have
in the
in the
show that
and 2.16
the enzyme
hour
ribosomes,
difficult
enzyme.
albeit
capacity
two rows,
of 1.7
intact
the eluted
by an enhancement
first
extracts
the
first
enzyme on the proliferative
amounted cells
into
stem cells,
associated
the small
was incorporated
with
during
While
has made it
associated
enzyme may enter
55%.
mitochondria,
fractions
of various
45% of the residual
(membranes,
remaining
subcellular
chromatography
labelled
the
125 I activity
of the 125 I label the
for
contained
material
dismutase ( q(o---o), RHS orError bars re-
superoxide bone marrow RHS ordinate).
the supematant
125 I activity
RESEARCH COMMUNICATIONS
for
effect donor
X-irradiation.
stem
Vol. 67, No. 3, 1975
Table
1.
BIOCHEMICAL
Prophylactic and oxide dismutase with respect to mice on day 10.
therapeutic effect of an intravenous dose of super(SOD) on X-irradiated donor bone marrow stem cells their ability to form spleen colonies (SC) in host Amount of SOD/injection = 35 ~~g/g. NO.
Time of treatment of donor
of spleen
0.9% saline
mice
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
colonies
SOD
(NS)
Mean + U
N
Radioprotective
(SC) formed N
effect
SC(SOIJ)
Mean + o
SC(NS)
-$ h before
350 rad
lh
350 350
"
1 h after
6
6.3 + 3.6
8
10.5
+ 2.7
1.7
"
25
9.8 + 3.3
32
21.3
+ 5.6
2.16
"
24
10
+ 5.4
23
14
+ 6.6
1.4
21
13.9
+ 8.0
26
35.0
+ 9.8
2.5
the
extent
1 h before and after 350 rad
The level it
of enzyme
excessive
nullify
its
35 pg/g
body weight,
a factor
+ 0.15
unity, It
unirradiated
+ 0.18,
appears cells,
(2 h) period,
did
proliferative
capacity
apparent
enzyme
is
able
while
and 0.7 + 0.1,
stem
of the
action
not
as indicated
of protection
to act
metabolically
largest
at JO and 100 rig/g
and suggests
to be related
to the irradiated to the
is,
turnover
of host
in principle,
intracellularly for
effect
functional
with mice
difficult by dismutating
being
at 100 pg/g state,
to
less
the
however,
of enzyme respect
for
the
enzyme since same
to their 2).
unless
At
was reduced
value,
(Table
3.
amounting
factor
that
same amount
show a deleterious spleens
this
dismutase
in Fig.
effect,
The latter
sensitization
exposed
superoxide
by the data
the
respectively.
in the
toxicity
The rapid
of exogenous
the enzyme elicited
signifies
toxic.
generated
quantities
radioprotective
of 2.16
to 1.15
is
seems to determine
affords. However,
than
therefore
An account the
superoxide
exogenous anions,
purposes.
of the enzyme
1171
in stem cells
(Fig.
Z),
along
Vol. 67, No. 3, 1975
BIOCHEMICAL
AND BIOPHYSICAL
SUPEROXIDE
DISMIJTASE
RESEARCH COMMUNICATIONS
(pg/g)
Figure 3. Radioprotective effect on bone marrow stem cells from donor mice, given variable amounts of bovine superoxide dismutase intravenously 1 h beError bars represent standard fore X-irradiation to 350 rad at 100 radfmin. deviations.
with
its
therapeutic
more effective extended
if
into
treated
given
injections
1 h later,
were
determined
by the comparative
row).
factor
enzyme when given
second
to possess
The increment
radioprotective
therapeutic
small
irradiated
mice
(details
2.16
with
the
the X-ray
dose of enzyme has also
exposure been
to be published
DISCUSSION
The capacity
development
of damage in the post-irradiation
it
occurs
water
well
radiolysis.
quirement
is
beyond
of superoxide
the lifetime
Since therefore
the imposed
assay
from
found
technique
to 2.5
first
as
(Table
of the
1, third
to increase
1, of the
effect
(Table
1 h
eluted
in the value
therapeutic
row).
the
A
survival
of
elsewhere). dismutase
enzyme reacts
to protect period
specifically
a secondary
1172
the
capacity,
of the short-lived
for
mice were
The stem cells,
proliferative
be
that
donor
of SOD at 35 ug/g;
colony
is consistent
an interval
Accordingly,
a better
spleen
the enzyme might
over
1 h after.
of 0.34
1 h after
that
doses
period.
dose and the found
suggested
in multiple
two intravenous
the X-ray
fourth
(4,5)
the post-irradiation
with
before
potential
supply
is
against
significant
primary with
the in that
radicals 0;
(6),
of the superoxide
from a re-
Vol. 67, No. 3, 1975
Table
2.
BIOCHEMICAL
Effect marrow mice.
of bovine stem cells
superoxide transplanted
No. of donor marrow Treatment donor
of
stem
injected
per
dismutase (SOD) on unirradiated into supralethally irradiated
(0.9%)
7, SOD 100 pip/g I! None
host
10th
spleens
anions
colonies
confluent
taken
as a measure
from which
related
to the
transfer chain-type
oxidations
26
105 + 37
2.25
o/30
30
96 + 23
9
O/27
27
116 + 28
2.25
o/30
30
105 + 31
0
63190
27
to the irradiated
if
not
might
oxide excess
sources,
and energy
in magnitude,
is
at critical
state,
production
to effect
in protective
anions
of a metabolic
aberrant
are
(9).
damage unless,
mechanisms
has occurred
and enhanced
are
aspects
of electron are,
and unirradiated irradiated
where
they
nature transfer in principle cells, cell,
or the production
to the point
of which
( 4, 7, 8) are but
the latter
in the
electron
molecules,
and more continuing
Since
irradiated
sources
in time,
of target
of
stem cells.
Potential
and enzyme cofactors
common to both
not be expected
breakdown
and radicals
95 + 13
of treated
limited
8
Mean weight
reliably.
capacity
and therefore
of sulfhydryls
but
in respiration
to count
78+
6
spleens
damage may proceed.
oxygen
Other
specific
of normal
of proliferative
a few examples. not
(nx)
1127
state
between
mice
9
post-irradiation
reactions
of spleens*Mean + 0
N
of host
and too numerous
irradiated
Weight
day
lethality
mouse
\Jeight
* Spleen
donor host
hone cells
(x lO+j)
mice
Saline
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
they a
of superaccumulate
in
sites.
The attraction
and collection
of 02 at membrane
1173
surfaces
has been
BIOCHEMICAL
Vol. 67, No. 3, 1975
noted
previously
(10)
damage by the oxygen the damage tend
to its
as has the radical
full
extent
for
many hours
into
superoxide
dismutase
(13).
superoxide
dismutase
provide
AND BIOPHYSICAL
susceptibility
or its
derivatives
in irradiated
the post-irradiation These
characteristics
a basis
for
RESEARCH COMMUNICATIONS
of membranes (11,
12).
to oxidative Development
membranes
is
slow
period
unless
and can exinhibited
of membrane
the therapeutic
of
effect
protection of the
by by enzyme
on stem cells.
REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13.
Petkau, A., Chelack, W. S., Pleskach, S. D., Meeker, B. E., and Brady, C. M. (1975) Biochem. Biophys. Res. Commun. 65, 886-893. J. E. (1960) Radiat. Res. 2, 115-125. McCulloch, E. A., and Till, Greenwood, F. C., Hunter, W. M., and Glover, J. S. (1963) Biochem. J. 89, 114-123. Biol. 6, Petkau, A., and Chelack, W. S., (1974) Int. J. Radiat. 421-426. Petkau, A. , Chelack, W. S., Pleskach, S. D., and Copps, T. P. (1975) Can. Fed. Biol. Sot. 18, 128. McCord, J. M. and Fridovich, I. (1969) J. Biol. Chem. 244, 6049-6055. R. U. (1970) Can. J. Chem. 8, 417-421. Packer, 3. E., and VJinchester, Bielski, B. H. J., and Chan, P. C. (1973) Archiv. Biochem. Biophys. 159, 873-879. Bors, W., Saran, M., Lengfelder, E., SpEttl, R., and Michel, C. (1974) Current Topics in Radiat. Res. Quarterly 2, 247-309. Petkau, A. (1971) Can. J. Chem. 9, 1187-1195. Pederson, T. C., and Aust, S. D. (1972) Biochem. Biophys. Res. Commun. ltg, 789-795. Tyler, D. D. (1975) FEBS Letters 51, 180-183. Petkau, A. and Chelack, W. S. (1974) Federat. Proceedings 33, 1505.
1174
BIOCHEMICAL
RADIOPROTECTION
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
OF BONE MARROW STEM CELLS BY SUPEROXIDE DISMUTASE
A. Petkau, K. Kelly', and B. E. Meeker
W. S. Chelack,
S. D. Pleskach,
C. Barefoot?,
Medical Biophysics Branch, Whiteshell Nuclear Research Establishment, Atomic Energy of Canada Limited, Pinawa, Manitoba, Canada ROE 1LO tImmunology Department, University of Manitoba, Winnipeg, Canada Received
October
16,1975
SUMMARY In mouse bone marrow cells the radioactivity from intravenously injected 125 I - superoxide dismutase reached its maximum level 1 h later. The 1 h interval was used while examining the effect of the unlabelled enzyme on the proliferative capacity of X-irradiated hematopoietic stem cells, as measured by When administered to donor mice their ability to form colonies in host spleens. in the amount of 35 ngfg body weight, the enzyme protected the cells, exposed to 350 rads, by a factor of 2.16 50.18. At doses of 15, 70, and 100 pg/g, this factor was reduced to 1.44 + 0.18, 1.15 t 0.15, and 0.7 + O.l,respectively. The value of 2.16 at 35 pg/g was increased to 2.5 by giving an additional therapeutic dose of the same size 1 h after the X-irradiation. INTRODUCTION
Recently,
it
was shown
superoxide
dismutase
protected
of ionizing
radiation
(1).
dose range
where
This
depression
of hematopoiesis
survival.
effect
of the enzyme on the blood
protect colony
of its
association
suggested
with
the proliferative assay
technique
white
protection
animal
terms
It
Swiss
that
the
present forming
bone marrow
capacity
of these
an intravenous mice
dose of bovine
from the
was elicited is study organ
the
effects
within
a radiation
principal
in which in mice
stem cells cells,
lethal
defect
governing
the radioprotective is
examined
and in its as measured
ability
both
in to
by the spleen
(2).
MATERIALS AND METHODS C3H/HeB inbred male mice weighing 16-22 grams each, were obtained from RioBreeding Laboratories of Canada Ltd., Ottawa, and from Canadian Breeding Farms and Laboratory Ltd., St. Constant, Quebec. Each shipment of mice, upon arrival, was divided randomly into donor and host groups and given food and water ad libitum, The host mice were housed in groups of 3 per cage. Lyophilized superoxide dismutase, obtained from Truett Laboratories, Dallas, Texas was dissolved in sterile normal saline (0.9%) for intravenous administration. In the solution, the enzyme concentration was 0.60, 1.4, 2.8, or 4.0 mgfml depending on whether a 20 gram donor mouse was to receive 15, 35, 70, or 100 ng of enzyme per gram body weight, respectively, in an injected (via tail vein) volume of 0.5 ml. Enzyme-treated donor mice of lesser or greater weight received proportionately smaller and larger volumes, respectively, Control donor mice were injected either 1 h before or 1167
Vol. 67, No. 3, 1975
1 h after
BIOCHEMICAL
X-irradiation
or at both
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
times
Fn tandem.
Donor mice were exposed to 350 rads, at a rate of 100 with 250 KV, 15 mA X-rays filtered by 0.5 mm Cu and 1 mm Al. received no prior treatment and were supralethally irradiated with X-rays of identical quality during the evening preceding which they were intravenously injected with donor bone marrow
rad/min, Host mice to 800 rads the day in stem cells.
One hour after completion of the irradiation or post-exposure treatment, the donor mice were sacrificed and the femora removed. The bone marrow stem cells from each femur were eluted as described previously (2) with 0.5 ml of Hank's balanced salts (HBS) solution. In any particular experiment, samples from individual mice in each treatment category were pooled and stored on ice prior to use. The number of nucleated cells in the pooled samples was counted in a hemocytoneter. Each sample was divided into two parts with adjustment in the cell number in one part to 18 x 106 and in the other part to 4.5 x 106 cells/ml, by dilution with HBS solution. The diluted cell suspensions were injected by tail vein into the host mice, with each mouse receiving 0.5 ml. Each dilution was injected into a minimum of 27 mice. The host mice were observed for 10 days during which some of them died. The surviving fraction was generally larger in groups that had received donor stem cells treated with superoxide dismutase rather than 0.9% saline. The survivors were sacrificed, the spleens removed and fixed in Bouin's fixative. The number of colonies on the surface of each spleen was counted with the aid of a low power (20X) stereomicroscope. In instances where the colonies were too numerous and confluent, the spleen weight was taken as an indicator of stem cell proliferative activity. During the course of this study the chloramine-T technique (3) was used to label the tyrosine residues of bovine superoxide dismutase with =51. The chemicals required in the preparation were bovine superoxide dismutase ((SOD), Truett Laboratories) and reagent grade chloramine-T, sodium metabisulfite, potassium iodide, and 0.05 M phosphate buffer, pH 7.5. Sodium (lz51) iodide was obtained from New England Nuclear Canada, Dorval, Quebec. It was supplied in aqueous solution, p Ii 8 - 10, and specified as thiosulfate-free as well as carrier-free, with an isotopic purity > 99%. The reagents were freshly prepared in the 0.05 M phosphate buffer to the following concentrations per ml: SOD 8 mg, chloramine-T 4 mg, Na2S205 2.4 mg, and KI 10 mg. To the reaction vial, containing 2 mCi of Na 1251/0.0045 ml, were added 0.025 ml each of 0.05 M phosphate buffer (pH 7.5), SOD, and chloramine-T solutions with brief mixing after each addition. Following the addition of chloramine-T, the reaction was allowed to proceed for 5 min before 0.1 ml of Na2S205 and 0.2 ml of KI were added. The labelled enzyme was separated from free 1251 on a Bio-gel P-60 column, equilibrated beforehand in 0.05 M phosphate buffer and presaturated with bovine serum albumin (25 mg/0.5 ml) to eliminate absorption of the labelled enzyme. the latter was collected in 0.5 ml fractions which were The labelled enzyme was pooled as indicated ~,"~?de:~:iY~~I activity. (Fig. 1) and further purified by exhaustive dialysis against physiological The labelled enzyme had an activity of 5 x 10' saline (0.15 M, pH 7.4). cpm/ml, equivalent to 3.4 x lo6 cpm/l.lg SOD. It was administered to mice in uptake studies in which the organ distribution and cellular uptake of the labelled enzyme could be followed. The small quantities (0.1 - 0.3 The mice were ml/mouse) of the 1251-SOD were injected intravenously. sacrificed at various times thereafter to collect serum and bone marrow stem cells for the purpose of counting their 1251 activity. RESULTS
The elution
is
in Fig.
given
profile
1 which
of the 125 I-labelling
shows
that
more than
1168
reaction 50% of the
mixture radionuclide
BIOCHEMICAL
Vol. 67, No. 3, 1975
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
IO-
I
2-
IO
also
on Rio-gel I-labelled
to the superoxide
dismutase
separated
f/15-40 used
were
from pooled
to obtain
mouse serum
the
and,
the
after
in-vivo
the amount
paralled
level
with
the eluted
eluted
its
drop
Removal
of the
remaining
with
in the
cells
former
70
00
data
W phosphate dismutase
labelled
the 125
of
physiological
of Fig.
2.
It
enzyme decreased
tubes saline,
shows
after
of eluted
The amount
to increase
that
half
suspensions
contained
by differential
the nucleated
cells.
during
both
the the
erythrocytes lysis
left
When the
1169
in an hour
bone marrow
directly
within
declined associated hour
first
first
four
41% of the were
125
lysed
in with
and was hours.
and nucleated
latter
buffer (1251-SOD)
enzyme was
I-SOD within
against
In suspensions
9c
to a maximum at 1 h and thereafter
appeared
total
bone marrow
and that
in the serum.
also
60
P-60 column, 0.05 bovine superoxide
dialysis
of labelled in 6 h.
40 50 NUMBER
Fractions
.
uptake
increased
cells
10 - 18% of the
125I
free
and was Q 90% cleared cells,
30 TUBE
.
Figure 1. E1utyy5yf;lf25 pH 7.5, of free
was bound
20
The cells.
I activity and centri-
BIOCHEMICAL
Vol. 67, No. 3, 1975
AND BIOPHYSICAL
Figure 2. Variation with time of 1251-labelled SOD) in serum (n ), LHS ordinate), eluted dinate), and bone marrow stem cells (X-X), present standard deviations.
fuged
(1 h at 1000,000
x g),
and the particulate
nuclei,
accounted
etc.)
radioactivity strate from
of the
that gel
the
is
The increase superoxide
dismutase
was also
accompanied
of the given
tissue
the
in Table
1,
with
to a factor
treated
with
one-half
times,
results
amount
before
that
of exogenous stem
cells
effect The data,
radioprotective
respectively,
30-70%
extent.
radioprotective
of the cells.
that
suggests
bone marrow
and one hour
1170
It
and low
to demon-
indicated
to a small
this
volume
SOD,preliminary have
in the
in the
show that
and 2.16
the enzyme
hour
ribosomes,
difficult
enzyme.
albeit
capacity
two rows,
of 1.7
intact
the eluted
by an enhancement
first
extracts
the
first
enzyme on the proliferative
amounted cells
into
stem cells,
associated
the small
was incorporated
with
during
While
has made it
associated
enzyme may enter
55%.
mitochondria,
fractions
of various
45% of the residual
(membranes,
remaining
subcellular
chromatography
labelled
the
125 I activity
of the 125 I label the
for
contained
material
dismutase ( q(o---o), RHS orError bars re-
superoxide bone marrow RHS ordinate).
the supematant
125 I activity
RESEARCH COMMUNICATIONS
for
effect donor
X-irradiation.
stem
Vol. 67, No. 3, 1975
Table
1.
BIOCHEMICAL
Prophylactic and oxide dismutase with respect to mice on day 10.
therapeutic effect of an intravenous dose of super(SOD) on X-irradiated donor bone marrow stem cells their ability to form spleen colonies (SC) in host Amount of SOD/injection = 35 ~~g/g. NO.
Time of treatment of donor
of spleen
0.9% saline
mice
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
colonies
SOD
(NS)
Mean + U
N
Radioprotective
(SC) formed N
effect
SC(SOIJ)
Mean + o
SC(NS)
-$ h before
350 rad
lh
350 350
"
1 h after
6
6.3 + 3.6
8
10.5
+ 2.7
1.7
"
25
9.8 + 3.3
32
21.3
+ 5.6
2.16
"
24
10
+ 5.4
23
14
+ 6.6
1.4
21
13.9
+ 8.0
26
35.0
+ 9.8
2.5
the
extent
1 h before and after 350 rad
The level it
of enzyme
excessive
nullify
its
35 pg/g
body weight,
a factor
+ 0.15
unity, It
unirradiated
+ 0.18,
appears cells,
(2 h) period,
did
proliferative
capacity
apparent
enzyme
is
able
while
and 0.7 + 0.1,
stem
of the
action
not
as indicated
of protection
to act
metabolically
largest
at JO and 100 rig/g
and suggests
to be related
to the irradiated to the
is,
turnover
of host
in principle,
intracellularly for
effect
functional
with mice
difficult by dismutating
being
at 100 pg/g state,
to
less
the
however,
of enzyme respect
for
the
enzyme since same
to their 2).
unless
At
was reduced
value,
(Table
3.
amounting
factor
that
same amount
show a deleterious spleens
this
dismutase
in Fig.
effect,
The latter
sensitization
exposed
superoxide
by the data
the
respectively.
in the
toxicity
The rapid
of exogenous
the enzyme elicited
signifies
toxic.
generated
quantities
radioprotective
of 2.16
to 1.15
is
seems to determine
affords. However,
than
therefore
An account the
superoxide
exogenous anions,
purposes.
of the enzyme
1171
in stem cells
(Fig.
Z),
along
Vol. 67, No. 3, 1975
BIOCHEMICAL
AND BIOPHYSICAL
SUPEROXIDE
DISMIJTASE
RESEARCH COMMUNICATIONS
(pg/g)
Figure 3. Radioprotective effect on bone marrow stem cells from donor mice, given variable amounts of bovine superoxide dismutase intravenously 1 h beError bars represent standard fore X-irradiation to 350 rad at 100 radfmin. deviations.
with
its
therapeutic
more effective extended
if
into
treated
given
injections
1 h later,
were
determined
by the comparative
row).
factor
enzyme when given
second
to possess
The increment
radioprotective
therapeutic
small
irradiated
mice
(details
2.16
with
the
the X-ray
dose of enzyme has also
exposure been
to be published
DISCUSSION
The capacity
development
of damage in the post-irradiation
it
occurs
water
well
radiolysis.
quirement
is
beyond
of superoxide
the lifetime
Since therefore
the imposed
assay
from
found
technique
to 2.5
first
as
(Table
of the
1, third
to increase
1, of the
effect
(Table
1 h
eluted
in the value
therapeutic
row).
the
A
survival
of
elsewhere). dismutase
enzyme reacts
to protect period
specifically
a secondary
1172
the
capacity,
of the short-lived
for
mice were
The stem cells,
proliferative
be
that
donor
of SOD at 35 ug/g;
colony
is consistent
an interval
Accordingly,
a better
spleen
the enzyme might
over
1 h after.
of 0.34
1 h after
that
doses
period.
dose and the found
suggested
in multiple
two intravenous
the X-ray
fourth
(4,5)
the post-irradiation
with
before
potential
supply
is
against
significant
primary with
the in that
radicals 0;
(6),
of the superoxide
from a re-
Vol. 67, No. 3, 1975
Table
2.
BIOCHEMICAL
Effect marrow mice.
of bovine stem cells
superoxide transplanted
No. of donor marrow Treatment donor
of
stem
injected
per
dismutase (SOD) on unirradiated into supralethally irradiated
(0.9%)
7, SOD 100 pip/g I! None
host
10th
spleens
anions
colonies
confluent
taken
as a measure
from which
related
to the
transfer chain-type
oxidations
26
105 + 37
2.25
o/30
30
96 + 23
9
O/27
27
116 + 28
2.25
o/30
30
105 + 31
0
63190
27
to the irradiated
if
not
might
oxide excess
sources,
and energy
in magnitude,
is
at critical
state,
production
to effect
in protective
anions
of a metabolic
aberrant
are
(9).
damage unless,
mechanisms
has occurred
and enhanced
are
aspects
of electron are,
and unirradiated irradiated
where
they
nature transfer in principle cells, cell,
or the production
to the point
of which
( 4, 7, 8) are but
the latter
in the
electron
molecules,
and more continuing
Since
irradiated
sources
in time,
of target
of
stem cells.
Potential
and enzyme cofactors
common to both
not be expected
breakdown
and radicals
95 + 13
of treated
limited
8
Mean weight
reliably.
capacity
and therefore
of sulfhydryls
but
in respiration
to count
78+
6
spleens
damage may proceed.
oxygen
Other
specific
of normal
of proliferative
a few examples. not
(nx)
1127
state
between
mice
9
post-irradiation
reactions
of spleens*Mean + 0
N
of host
and too numerous
irradiated
Weight
day
lethality
mouse
\Jeight
* Spleen
donor host
hone cells
(x lO+j)
mice
Saline
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
they a
of superaccumulate
in
sites.
The attraction
and collection
of 02 at membrane
1173
surfaces
has been
BIOCHEMICAL
Vol. 67, No. 3, 1975
noted
previously
(10)
damage by the oxygen the damage tend
to its
as has the radical
full
extent
for
many hours
into
superoxide
dismutase
(13).
superoxide
dismutase
provide
AND BIOPHYSICAL
susceptibility
or its
derivatives
in irradiated
the post-irradiation These
characteristics
a basis
for
RESEARCH COMMUNICATIONS
of membranes (11,
12).
to oxidative Development
membranes
is
slow
period
unless
and can exinhibited
of membrane
the therapeutic
of
effect
protection of the
by by enzyme
on stem cells.
REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13.
Petkau, A., Chelack, W. S., Pleskach, S. D., Meeker, B. E., and Brady, C. M. (1975) Biochem. Biophys. Res. Commun. 65, 886-893. J. E. (1960) Radiat. Res. 2, 115-125. McCulloch, E. A., and Till, Greenwood, F. C., Hunter, W. M., and Glover, J. S. (1963) Biochem. J. 89, 114-123. Biol. 6, Petkau, A., and Chelack, W. S., (1974) Int. J. Radiat. 421-426. Petkau, A. , Chelack, W. S., Pleskach, S. D., and Copps, T. P. (1975) Can. Fed. Biol. Sot. 18, 128. McCord, J. M. and Fridovich, I. (1969) J. Biol. Chem. 244, 6049-6055. R. U. (1970) Can. J. Chem. 8, 417-421. Packer, 3. E., and VJinchester, Bielski, B. H. J., and Chan, P. C. (1973) Archiv. Biochem. Biophys. 159, 873-879. Bors, W., Saran, M., Lengfelder, E., SpEttl, R., and Michel, C. (1974) Current Topics in Radiat. Res. Quarterly 2, 247-309. Petkau, A. (1971) Can. J. Chem. 9, 1187-1195. Pederson, T. C., and Aust, S. D. (1972) Biochem. Biophys. Res. Commun. ltg, 789-795. Tyler, D. D. (1975) FEBS Letters 51, 180-183. Petkau, A. and Chelack, W. S. (1974) Federat. Proceedings 33, 1505.
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