- Email: [email protected]
Estrogen regulation of superoxide dismutase in normal rat mammary tissues and mammary tumors
Vol. 113, No. 3, 1983
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS Pages 883-887
June 29, 1983
ESTROGEN REGULATION OF SUPEROXIDE DISMUTASE IN NORMAL RAT MAMMARY TISSUES AND MAMMARY TUMORS C. Whiteside,
R,H,
Department
Received
April
14,
Blackman,
and T.A.
Bremner
of Zoology, Howard University Washington, D.C. 20059
1983
SUMMARY. Multiple electrophoretic molecular variants of superoxide dismutase were demonstrated in normal rat mammary tissues and DMBA-induced rat mammary tumors, The specific activities of Cu/Zu superoxide dismutase in mammary tumors of estrogen-treated rats were not significantly different from those activities seen in normal rat mammary tissues. However, the enzyme activities of mammary tumors from untreated rats (no estrogen) were significantly lower than the activities of normal rat mammary tissues. Exogenous estrogen appeared to raise superoxide dismutase levels in DMBA-induced rat mammary tumors to those levels seen in normal rat mammary tissues.
Many biochemical of superoxide
radicals
These
radicals,
cause
of oxidative
acts
it
hydrogen
Reduced animal
tissues
are
metabolic
similar
the
cells
univalent
in the
cell
these
anions
are
(1,2). the
lead
to the
reduction
to accumulate,
enzyme against
of oxygen
believed Superoxide
toxic
effects
by catalyzing
production (1).
to be the
major
dismutase
(SOD)
of superoxide
their
conversion
to
(3).
SOD levels
undifferentiated
levels
damage
scavenges
peroxide
in aerobic
through
when allowed
as a protective
radicals;
cells
reactions
have generally
(.4,5,6).
Similar
cells
(4,7).
findings Since
in many aspects
states
then
of cancer
cells
it
been demonstrated
like
been reported
undifferentiated
including
seems reasonable should,
have also
their
cells growth
to hypothesize
undifferentiated
ABBREVIATION: SOD, superoxide dismutase; anthracene; EDTA, ethylenediaminetetraacetic tetrazolium.
in transformed
cells,
and cancer patterns
and
that
SOD
the
be lower
than
DMBA, 7,12-dimethylbenz(a)acid; NBT, nitroblue
0006-291 883
for
Copyright AN rights of
X/83
$1 .50
0 1983 by Academic Press, Inc. reproduction in lrny form reserved.
Vol. 113, No. 3, 1983
those
of normal
length
cells.
by Oberley Cell
growth
action
of estrogen actions
Numerous transformation as opposed formation
treated
also
are display
on DMBA-induced
is discussed
at
rat
influenced
by hormones;
and
(9).
The
hormone-sensitivity
mammary tumors
exemplifies
a relationship
between
such
(10,ll). studies (8).
have established However,
to SOD isozymes.
most
of these
of SOD expression
(no estrogen) in transformed
MATERIALS
emphasize
SOD isozyme
SOD activities
and DMBA-induced
rats
studies
We investigated
and compare
mammary tissues
and untreated
mechanisms
RESEARCH COMMUNICATIONS
of SOD in neoplasia
differentiation
of some tumors
to determine rat
The role
and cell
patterns
hormonal
AND BIOPHYSICAL
(8).
growth
normal
BIOCHEMICAL
rat
SOD activities
variation
and isozyme mammary tumors
to elucidate
SOD and
genetic
in trans-
patterns
of
of estrogenregulatory
tissues.
AND METHODS
Fifty-day old female Sprague-Dawley rats were injected with 7,12dimethylbenz(a)anthracene (DMBA, 10 mg/ml peanut oil). After development of palpable tumors, animals were given 17-B estradiol pellets (15 mg/ pellet) subcutaneously. Another group of tumor bearing animals did not receive estrogen treatment. The animals were sacrificed three days later and tumors were removed, Control animals were injected with 5 ug diethylstilbestrol to promote mammary tissue growth. Normal mammary tissues and mammary tumors were homogenized with equal volumes of 0.2M KH2P04, pH 7.4; 1.0 mM dithiothreitol; 1.0 mM EDTA; O.OlM isoascorbic acid and 5% glycerol. The homogenate was centrifuged at 1,000 The x g for 15 minutes at 4 C to remove any unbroken cells and nuclei. supernatant was centrifuged at 30,000 x g for one hour at 4 C to obtain a cytosolic fraction. Any contaminating hemoglobin was removed by chloroform-ethanol extraction (12). SOD was contained in the upper phase. SOD was assayed according to the method of Beauchamp and Fridovich (13) with modifications, A microassay was performed using a 24-well COSTAR tissue culture plate. This plate was used instead of test tubes and assured direct exposure of the reaction mixture to light. The reaction mixture contained 0.075 mM nitroblue tetrazolium (NBT), O.OlM EDTA/O.O3 mM NaCN, 0.006 mM riboflavin, the desired quantity of enzyme and 0.0536M KH2P04, pH 7.8 to make a total volume of 1.0 ml. Each well was exposed to light for 8 minutes after the addition of riboflavin and the absorbance was measured at 660 nm. One unit of enzyme activity is defined as that amount of enzyme causing half maximum inhibition of NBT reduction. Molecular variants of the enzyme were detected by polyacrylamide slab gel electrophoresis using a 12% polyacrylamide gel (14) without sodium dodecyl sulfate. Staining of the gels was achieved by incubation for 20 minutes in the dark in a reaction mixture containing 2.0 ml of 0.0122M NBT, 8.3 ml of 0.0078M phenazine methosulfate, 25 ml of 0.2M Tris, pH 8.0 884
BIOCHEMICAL
Vol. 113, No. 3, 1983
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
and distilled water to a final volume of 100 ml followed by illumination with a cool white fluorescent lamp (15 watts) until bands were visible. Bands appear white against a dark blue background. Tissue samples were applied to a second gel, electrophoresed and subsequently stained as previously described after a 20 minute incubation in an 8 mM NaCN solution. Protein concentrations were determined according to the method of Bradford (15). The Student's t-test was used to determine of differences between group means.
statistical
significance
RESULTS AND DISCUSSION No significant
difference
SOD of mammary tumors tissues.
However,
seen in normal administered levels
reduced
mary tumors variants with
were
of the
patterns
of the
: 2005 F Y ” ‘00 GI G k! v) ’
Fig.
1
with
(fig.
in the
estrogen
1).
rat
effect
of
mammary
SOD activities
and those
We hypothesize
a corrective
of normal 2). present.
of pre-treatment
which
and normal
activities
activities that
by raising
the SOD
state.
(fig.
enzyme were
specific
difference
treated
exhibiting
tumorous
similar
the exception
to determine
is
in the
The SOD isozyme
not
mammary tissues
estrogen
rats
was a significant
from rats
rats
in the
from estrogen-treated
there
of mammary tumors
was observed
bands were
rat
In both
mammary tissues
and rat
cases,
molecular
A second with
gel,
multiple identical
8 mM NaCN solution,
cyanide
sens itive,
mam-
to the
first
was stained
a character
istic
T
NORMAL
OMBA
ONLY
DMBA
& E2
Distribution of the Cu/Zn SOD activities (+ S.D.) in normal mammary tissues and DMBA-induced rat mammary tumors from estrogen (Ez)-treated and untreated (no EZ) rats. 885
rat
of
Vol. 113, No. 3, 1983
BIOCHEMICAL
---
-----
02
1
3
4
5
RESEARCH COMMUNICATIONS
8
123
4
5
Fig.
2
Isozyme patterns on polyacrylamide gel electrophoretograms of superoxide dismutase. Lanes 1, 2, 3 contained tissue samples (150 ug protein) of normal rat mamnary tissues. Lanes 4, 5, 6 contained tissue samples (150 ug protein) of rat mammary tumors from estrogen-treated rats. Band width approximates the amount of enzyme activity.
Fig.
3
Isozyme patterns on polyacrylamide gel electrophoretograms of superoxide dismutase subsequent to incubation in 8mM NaCN solution.Lanes 1, 2, 3 contained tissue samples (150 ug protein) of normal rat mammary tissues. Lanes 4, 5, 6 contained tissue samples (150 ug protein) of rat mammary tumors from estrogentreated rats. Band width approximates the amount of enzyme activity.
CulZn
SOD.
sumably
estrogen
One
the The
The
2
AND BIOPHYSICAL
of
Cu/Zn
form
present on
possibility
the
the
three as
studies
well give
expression exists
major
all
SOD
this
in
seen
of
evidence
of that
as
bands
of
the an
in
the
minor
first bands
inductive
DMBA-induced
relationship
gel (fig.
effect rat
is
encompasses
all
pre-
3). of
mammary
6
exogenous
tumors. neoplastic
tissues.
ACKNOWLEDGEMENTS: Animals A. Anderson (Zoology Dept, by grant GM-07799 and grant
used in this Howard Univ.). 5-S06-RR-08016-11
study This
were supplied study was (DRR/NIH)
by Dr. supported
Winston in part
REFERENCES 1.
2. 3.
Fridoyich, Pridovich, McCord,
I1 I. J.M.
(1974) (1973) and I.
Adyances Biochem, Eridoyich
in Enzymology 41, 36-97, Sot, Trans. 1, 48-50, (1968) J. Biol, Chem. 243, 886
5753-5760.
-t
Vol. 113, No. 3, 1983
4. 5. 6. 7.
8. 9.
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
Bize, I.B., L.W. Oberley and H.P. Morris (1980) Cancer Res. 40, 36863693. Sahu, S.K., L,W. Oberley, R.H. Stevens and E,F, Rieley (1977) J. Nat'1 Cancer Inst, 58(4), 1125-1128. Dionisi, O., T. Galeotti, T. Terranova and A, Azzi (1975) Biochim. Biophys. Acta 403, 292-300, (.1976) Pediat. Res. 10, 154-158. Autor, A., L. Grand and R.J. Roberts (1980) Med. Hypoth. 6, Oberley, L.W., T.D. Oberley and G,R. Buettner 249-267. Huggins, C., L. Grand and F. Brillantes (1961) Nature (London) 189,
204-207. 10. 11. 12. :43: 15.
Shafie, S.M. (1980) Science 209, 701-702. Teller, M.N., C.C. Stock, G. Stohr, P.C. Marker, R.J. Kaufman, G.C. Escher and M. Bowie (1966) Cancer Res. 26(l), 245-252. Tsuchihashi, M. (1923) Biochemi Z. 140, 63-112. Beauchamp, C. and I. Fridovich (1971) Anal. Biochem. 44, 276-287. Laemmli, U.K. (1970) Nature 227, 680-685. Bradford, M. (1976) Anal. Biochem. 72, 248-254.
887
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS Pages 883-887
June 29, 1983
ESTROGEN REGULATION OF SUPEROXIDE DISMUTASE IN NORMAL RAT MAMMARY TISSUES AND MAMMARY TUMORS C. Whiteside,
R,H,
Department
Received
April
14,
Blackman,
and T.A.
Bremner
of Zoology, Howard University Washington, D.C. 20059
1983
SUMMARY. Multiple electrophoretic molecular variants of superoxide dismutase were demonstrated in normal rat mammary tissues and DMBA-induced rat mammary tumors, The specific activities of Cu/Zu superoxide dismutase in mammary tumors of estrogen-treated rats were not significantly different from those activities seen in normal rat mammary tissues. However, the enzyme activities of mammary tumors from untreated rats (no estrogen) were significantly lower than the activities of normal rat mammary tissues. Exogenous estrogen appeared to raise superoxide dismutase levels in DMBA-induced rat mammary tumors to those levels seen in normal rat mammary tissues.
Many biochemical of superoxide
radicals
These
radicals,
cause
of oxidative
acts
it
hydrogen
Reduced animal
tissues
are
metabolic
similar
the
cells
univalent
in the
cell
these
anions
are
(1,2). the
lead
to the
reduction
to accumulate,
enzyme against
of oxygen
believed Superoxide
toxic
effects
by catalyzing
production (1).
to be the
major
dismutase
(SOD)
of superoxide
their
conversion
to
(3).
SOD levels
undifferentiated
levels
damage
scavenges
peroxide
in aerobic
through
when allowed
as a protective
radicals;
cells
reactions
have generally
(.4,5,6).
Similar
cells
(4,7).
findings Since
in many aspects
states
then
of cancer
cells
it
been demonstrated
like
been reported
undifferentiated
including
seems reasonable should,
have also
their
cells growth
to hypothesize
undifferentiated
ABBREVIATION: SOD, superoxide dismutase; anthracene; EDTA, ethylenediaminetetraacetic tetrazolium.
in transformed
cells,
and cancer patterns
and
that
SOD
the
be lower
than
DMBA, 7,12-dimethylbenz(a)acid; NBT, nitroblue
0006-291 883
for
Copyright AN rights of
X/83
$1 .50
0 1983 by Academic Press, Inc. reproduction in lrny form reserved.
Vol. 113, No. 3, 1983
those
of normal
length
cells.
by Oberley Cell
growth
action
of estrogen actions
Numerous transformation as opposed formation
treated
also
are display
on DMBA-induced
is discussed
at
rat
influenced
by hormones;
and
(9).
The
hormone-sensitivity
mammary tumors
exemplifies
a relationship
between
such
(10,ll). studies (8).
have established However,
to SOD isozymes.
most
of these
of SOD expression
(no estrogen) in transformed
MATERIALS
emphasize
SOD isozyme
SOD activities
and DMBA-induced
rats
studies
We investigated
and compare
mammary tissues
and untreated
mechanisms
RESEARCH COMMUNICATIONS
of SOD in neoplasia
differentiation
of some tumors
to determine rat
The role
and cell
patterns
hormonal
AND BIOPHYSICAL
(8).
growth
normal
BIOCHEMICAL
rat
SOD activities
variation
and isozyme mammary tumors
to elucidate
SOD and
genetic
in trans-
patterns
of
of estrogenregulatory
tissues.
AND METHODS
Fifty-day old female Sprague-Dawley rats were injected with 7,12dimethylbenz(a)anthracene (DMBA, 10 mg/ml peanut oil). After development of palpable tumors, animals were given 17-B estradiol pellets (15 mg/ pellet) subcutaneously. Another group of tumor bearing animals did not receive estrogen treatment. The animals were sacrificed three days later and tumors were removed, Control animals were injected with 5 ug diethylstilbestrol to promote mammary tissue growth. Normal mammary tissues and mammary tumors were homogenized with equal volumes of 0.2M KH2P04, pH 7.4; 1.0 mM dithiothreitol; 1.0 mM EDTA; O.OlM isoascorbic acid and 5% glycerol. The homogenate was centrifuged at 1,000 The x g for 15 minutes at 4 C to remove any unbroken cells and nuclei. supernatant was centrifuged at 30,000 x g for one hour at 4 C to obtain a cytosolic fraction. Any contaminating hemoglobin was removed by chloroform-ethanol extraction (12). SOD was contained in the upper phase. SOD was assayed according to the method of Beauchamp and Fridovich (13) with modifications, A microassay was performed using a 24-well COSTAR tissue culture plate. This plate was used instead of test tubes and assured direct exposure of the reaction mixture to light. The reaction mixture contained 0.075 mM nitroblue tetrazolium (NBT), O.OlM EDTA/O.O3 mM NaCN, 0.006 mM riboflavin, the desired quantity of enzyme and 0.0536M KH2P04, pH 7.8 to make a total volume of 1.0 ml. Each well was exposed to light for 8 minutes after the addition of riboflavin and the absorbance was measured at 660 nm. One unit of enzyme activity is defined as that amount of enzyme causing half maximum inhibition of NBT reduction. Molecular variants of the enzyme were detected by polyacrylamide slab gel electrophoresis using a 12% polyacrylamide gel (14) without sodium dodecyl sulfate. Staining of the gels was achieved by incubation for 20 minutes in the dark in a reaction mixture containing 2.0 ml of 0.0122M NBT, 8.3 ml of 0.0078M phenazine methosulfate, 25 ml of 0.2M Tris, pH 8.0 884
BIOCHEMICAL
Vol. 113, No. 3, 1983
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
and distilled water to a final volume of 100 ml followed by illumination with a cool white fluorescent lamp (15 watts) until bands were visible. Bands appear white against a dark blue background. Tissue samples were applied to a second gel, electrophoresed and subsequently stained as previously described after a 20 minute incubation in an 8 mM NaCN solution. Protein concentrations were determined according to the method of Bradford (15). The Student's t-test was used to determine of differences between group means.
statistical
significance
RESULTS AND DISCUSSION No significant
difference
SOD of mammary tumors tissues.
However,
seen in normal administered levels
reduced
mary tumors variants with
were
of the
patterns
of the
: 2005 F Y ” ‘00 GI G k! v) ’
Fig.
1
with
(fig.
in the
estrogen
1).
rat
effect
of
mammary
SOD activities
and those
We hypothesize
a corrective
of normal 2). present.
of pre-treatment
which
and normal
activities
activities that
by raising
the SOD
state.
(fig.
enzyme were
specific
difference
treated
exhibiting
tumorous
similar
the exception
to determine
is
in the
The SOD isozyme
not
mammary tissues
estrogen
rats
was a significant
from rats
rats
in the
from estrogen-treated
there
of mammary tumors
was observed
bands were
rat
In both
mammary tissues
and rat
cases,
molecular
A second with
gel,
multiple identical
8 mM NaCN solution,
cyanide
sens itive,
mam-
to the
first
was stained
a character
istic
T
NORMAL
OMBA
ONLY
DMBA
& E2
Distribution of the Cu/Zn SOD activities (+ S.D.) in normal mammary tissues and DMBA-induced rat mammary tumors from estrogen (Ez)-treated and untreated (no EZ) rats. 885
rat
of
Vol. 113, No. 3, 1983
BIOCHEMICAL
---
-----
02
1
3
4
5
RESEARCH COMMUNICATIONS
8
123
4
5
Fig.
2
Isozyme patterns on polyacrylamide gel electrophoretograms of superoxide dismutase. Lanes 1, 2, 3 contained tissue samples (150 ug protein) of normal rat mamnary tissues. Lanes 4, 5, 6 contained tissue samples (150 ug protein) of rat mammary tumors from estrogen-treated rats. Band width approximates the amount of enzyme activity.
Fig.
3
Isozyme patterns on polyacrylamide gel electrophoretograms of superoxide dismutase subsequent to incubation in 8mM NaCN solution.Lanes 1, 2, 3 contained tissue samples (150 ug protein) of normal rat mammary tissues. Lanes 4, 5, 6 contained tissue samples (150 ug protein) of rat mammary tumors from estrogentreated rats. Band width approximates the amount of enzyme activity.
CulZn
SOD.
sumably
estrogen
One
the The
The
2
AND BIOPHYSICAL
of
Cu/Zn
form
present on
possibility
the
the
three as
studies
well give
expression exists
major
all
SOD
this
in
seen
of
evidence
of that
as
bands
of
the an
in
the
minor
first bands
inductive
DMBA-induced
relationship
gel (fig.
effect rat
is
encompasses
all
pre-
3). of
mammary
6
exogenous
tumors. neoplastic
tissues.
ACKNOWLEDGEMENTS: Animals A. Anderson (Zoology Dept, by grant GM-07799 and grant
used in this Howard Univ.). 5-S06-RR-08016-11
study This
were supplied study was (DRR/NIH)
by Dr. supported
Winston in part
REFERENCES 1.
2. 3.
Fridoyich, Pridovich, McCord,
I1 I. J.M.
(1974) (1973) and I.
Adyances Biochem, Eridoyich
in Enzymology 41, 36-97, Sot, Trans. 1, 48-50, (1968) J. Biol, Chem. 243, 886
5753-5760.
-t
Vol. 113, No. 3, 1983
4. 5. 6. 7.
8. 9.
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
Bize, I.B., L.W. Oberley and H.P. Morris (1980) Cancer Res. 40, 36863693. Sahu, S.K., L,W. Oberley, R.H. Stevens and E,F, Rieley (1977) J. Nat'1 Cancer Inst, 58(4), 1125-1128. Dionisi, O., T. Galeotti, T. Terranova and A, Azzi (1975) Biochim. Biophys. Acta 403, 292-300, (.1976) Pediat. Res. 10, 154-158. Autor, A., L. Grand and R.J. Roberts (1980) Med. Hypoth. 6, Oberley, L.W., T.D. Oberley and G,R. Buettner 249-267. Huggins, C., L. Grand and F. Brillantes (1961) Nature (London) 189,
204-207. 10. 11. 12. :43: 15.
Shafie, S.M. (1980) Science 209, 701-702. Teller, M.N., C.C. Stock, G. Stohr, P.C. Marker, R.J. Kaufman, G.C. Escher and M. Bowie (1966) Cancer Res. 26(l), 245-252. Tsuchihashi, M. (1923) Biochemi Z. 140, 63-112. Beauchamp, C. and I. Fridovich (1971) Anal. Biochem. 44, 276-287. Laemmli, U.K. (1970) Nature 227, 680-685. Bradford, M. (1976) Anal. Biochem. 72, 248-254.
887