Download Liver and Blood Cell Catatase Activity of Tumor

[CANCER RESEARCH 32, 1190-1194,
June 1972]
Liver and Blood Cell Catatase Activity of Tumor-bearing Mice
Joel H. Kaplan and James N. Groves
Life Sciences Branch, Corporate Research and Development Center, General Electric Company, Schenectady, New York 12301
SUMMARY
Liver and blood cell catalase activity in mice with and
without tumors of various sizes and origins was measured.
Animals bearing tumors >1.5 cm in size showed depression of
leukocyte and/or liver catalase activity when compared with
tumor-free animals, but this effect was not significant in mice
with smaller tumors. No depression of erythrocyte catalase
activity was observed.
INTRODUCTION
One of the earliest reports on the systemic effects of tumors
was made by Brahn (2), who found that the liver catalase
activity of human beings with cancer of the rectum, stomach,
pancreas, and intestine was very much lower than normal. This
systemic phenomenon
later was studied extensively by
Greenstein et al. (4-8) with the use of a large number and
variety of tumor-bearing mice and rats. However, no effect of
a growing tumor on erythrocyte catalase was observed (6).
These investigators (6, 8) postulated the existence of a toxic
material from the tumor which might be responsible for
lowering liver catalase activity. In later work, Nakahara and
Fukuoka (15) isolated a material from human cancer material
which they referred to as toxohormone. This material, when
injected into mice, brought about a lowering in the liver
catalase activity; it did not affect liver catalase in vitro (14).
More recent studies have shown that the behavior of
leukocyte catalase is similar to that of liver, rather than to that
of erythrocyte catalase. Rechcigl and Evans (17) reported that
a relatively
specific
inhibitor
of catalase
activity,
3-amino-l ,2,4-triazole, inhibited catalase in liver, kidney and
leukocytes but not in erythrocytes. In vitro experiments with
tumor extracts, which are known to depress the catalase
activity of liver but not of erythrocytes. also have been shown
to inactivate leukocyte catalase (3). We therefore asked
whether tumors in vivo have an effect on leukocyte catalase
activity similar to their effect on liver catalase activity.
Consequently, we have made a study of both liver and blood
cell catalase activity of inbred mice bearing both spontaneous
and transplanted tumors.
MATERIALS
AND METHODS
Animals and Tumors. Inbred strains of female mice were
used throughout. The strains and tumors used were: (a)
C3H/HeJ, bearing either spontaneous mammary tumor or
Received November 11, 1971 ¡acceptedMarch 6, 1972.
1190
transplanted lymphosarcoma 6C3HED; (b) DBA/1J, bearing
transplanted pleomorphic sarcoma S37; and (c) A/J, bearing
transplanted spindle-cell sarcoma Sal. Tumor-free animals of
each strain were used as controls. All mice were housed and
sacrificed at The Jackson Laboratory, Bar Harbor, Maine.
Blood
and
Liver Samples.
Blood samples
from
C02 -anesthetized mice were obtained by cardiac puncture and
diluted in 1.5 volumes of 0.9% NaCl solution and 1% heparin.
Blood was stored and shipped at 4°.Livers were excised from
each animal, quickly frozen in liquid nitrogen, and shipped in
Dry Ice.
Isolation of Blood Cell Fractions. Ficoll (Pharmacia Fine
Chemicals, Inc., Piscataway, N. J.), a sucrose polymer with a
molecular weight of about 400,000, was dialyzed to remove
the NaCl. Ficoll was made up as a 15% (w/w) aqueous solution
and was dialyzed against distilled water for 3 days at 3°.The
dialyzed preparation was filtered through a 0.45-^m Millipore
filter and then was subjected to concentration by lyophilization. To obtain the desired concentrations, we dissolved the
concentrated Ficoll in the appropriate volume of Seligmann's
balanced salt solution.1
Total leukocytes were isolated from blood samples received
24 hr after sacrifice of animals. To 2.0 ml of each blood
sample were added 150 p\ of a l%saponin solution containing
0.05 M KH2P04 in 0.9% NaCl solution, pH 7.5. The
saponin-treated blood was incubated for 2 min at 37°to lyse
the erythrocytes. The resulting hemolysate of each sample was
layered on top of 8 ml of a 13% (w/w) Ficoll solution and
centrifuged at 4°in a Lourdes Model 1201 swinging bucket
rotor (Lourdes Instrument Co., Old Bethpage, N. Y.) for 34
min at 413 X g. After centrifugation, the leukocytes could be
found at the bottom of the tube. Left behind, on top of the
13% (w/w) Ficoll solution, were platelets, red-cell ghosts,
plasma, and proteins-hemoglobin
being in the majority. The
pelleted leukocytes were washed in 3 volumes of Seligmann's
balanced salt solution and centrifuged at 250 X g for 30 min.
They were resuspended in 7.0 ml of 0.05 M KH2P04 . in 0.9%
NaCl solution, pH 7.5, and were used directly for
measurement of catalase activity. Cells were counted with a
Neubauer hemocytometer. The recovery of total leukocytes
was 12 ±2% which, although low, yielded a more than
adequate number of cells for determination
of catalase
activity.
We isolated erythrocytes by washing whole blood in 100
volumes of 0.05 M KH2P04 in 0.9% NaCl solution, pH 7.5.
'Comprised of 7.65 g NaCl, 0.20 g KC1, 1.50 g NaCH3CO2, 0.05 g
NaH7PO4, 0.10 g KH2PO4, 0.70 g NaHCO3, l g glucose, 0.003 g
ascorbic acid, and twice-distilled water to 1000 ml (pH of the solution,
7.3).
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Catatase Activity of Tumor-bearing Mice
The red cells were then resuspended in the same buffer and
used for catalase assay.
Liver Homogenates. Frozen livers were thawed at room
temperature, rinsed in twice-distilled water, and blotted dry.
Next, they were minced in 4 volumes of cold 7 mM phosphate
buffer, pH 7.2, containing 1 mM dithiothreitol (Cleland's
reagent) and then were homogenized in a Potter-Elvehjem
homogenizer by 10 strokes with a Teflon pestle. The
homogenates were centrifuged in the cold for 30 min at
48,000 X g. and the supernatants were drawn off and used for
catalase assay.
In some experiments, the homogenates were sonically
disrupted for 5 min at 0-5° (Branson 185 C, 20-kcps
ultrasonic disintegrator, set to deliver 65 watts at the tip of
the probe). The sonically disrupted homogenates were then
centrifuged in the cold for 30 min at 48,000 X g, and the
supernatants were used for measuring catalase activity. Protein
concentrations were determined by the method of Lowry et
al (12).
Catalase Assays. The main procedure for assay of catalase
activity involved the use of an instrument developed by one of
us at the General Electric Research and Development Center
(9). The instrumentation,
which has been thoroughly
described (9), is based on the use of a polypropylene
membrane-covered oxygen electrode communicating with a
reaction chamber in which the catalase-hydrogen peroxide
reaction is carried out. This system measures the oxygen
generation rate on a continuous linear basis which is directly
related to catalase activity. With the use of such an apparatus,
a SCU2 is defined as the number of /¿molesof hydrogen
peroxide decomposed per min per ml of reaction volume
under specified measurement conditions, namely, at 0.06 M
hydrogen peroxide and 37°.unless otherwise noted. This assay
procedure was used to obtain all data reported in Tables 1,2,
and 3 and will be referred to as the ''Groves assay." Unlike
some other assays for tissue catalase, which measure the
disappearance of hydrogen peroxide (14, 16) and thus also will
include measurement of a peroxidase reaction, this procedure,
by following a linear oxygen generation rate, measures only a
catalase reaction (9).
In some experiments (Chart 1), liver catalase activity on the
same sample also was determined by a procedure which was
essentially that described by Beers and Sizer (1). In this
procedure, the disappearance of hydrogen peroxide is followed
spectrophotometrically
at 240 nm. An approximately 0.05 M
solution of hydrogen peroxide substrate was prepared by fresh
dilution of 0.15 ml of 30% hydrogen peroxide (Superoxol;
Merck, Sharp and Dohme, Inc., Philadelphia, Pa.) with 25 ml
of 0.05 M KH2P04 buffer, pH 7.5, in 0.9% NaCl solution. A
silica cuvet in the optical path of a Beckman DU recording
spectrophotometer
served as the reaction vessel. The control
cuvet, containing 1.0 ml of hydrogen peroxide substrate and
2.0 ml of water, had an absorbance of 0.800 at 240 nm. The
reaction cuvet contained 2.0 ml of diluted liver supernatant.
At zero time, 1.0 ml of hydrogen peroxide substrate was
rapidly pipetted into the reaction cuvet, and the decrease of
absorbance at 240 nm was recorded for 70 sec. Duplicate
! The abbreviation used is: SCU, standard catalase activity unit.
140
120
100
? 80
60
40
20
20
40
60
80
100
120
140
BEERS ANOSIZER ASSAY
Chart 1. Correlation of the assay of Beers and Sizer with that of
Groves for liver catalase activity. The »,single measurements of liver
catalase activities of tumor-free and tumor-bearing mice. The
magnitudes of the horizontal and vertical bar lines about each point
were calculated from the coefficient of variations which describe the
precision of the Beers and Sizer assay and of the Groves assay,
respectively. The strains and tumors used were: (a) C3H/HeJ- 7 to
8-month-old mice bearing large (15- to 20 mm in size) spontaneous
mammary tumors; (ft) C3H/HeJ 2-month-old mice bearing transplanted
lymphosarcoma 6C3HED; and (e) DBA/1J 2-month-old mice bearing
transplanted pleomorphic sarcoma S-37. All measurements were made
on liver homogenates, except for tumor-free and tumor-bearing
C3H/HeJ 7- to 8-month-old mice, in which case catalase activities were
measured on both liver homogenates and sonically disrupted
homogenates. Straight line, calculated, least-square linear plot fit for
the experimental points. The activity values obtained from the Groves
assay should be multiplied by 1000 and those from the Beers and Sizer
assay should be multiplied by 100.
reactions were run on all liver samples. The absorbance
measurements were made at room temperature.
The disappearance of hydrogen peroxide may be described
by the equation for Ist-order reaction kinetics:
Log, o A = log! o A0 - to/2.3
where A0 is the absorbance due to hydrogen peroxide at 0
time, A is the absorbance due to hydrogen peroxide at time r,
and k is the rate constant. By plotting the logarithm of the
absorbance against time, one can calculate k from the slope
of the resulting straight line. Thus each catalase unit specifies
the relative logarithmic disappearance of hydrogen peroxide
per sec and is expressed per mg of liver protein.
RESULTS
The 1st series of experiments were performed on the blood
and liver of spontaneous mammary tumor-bearing C3H/HeJ
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1191
Joel H. Kaplan and James N. Groves
mice, 7 to 8 months of age. The average tumor size was 6.5
mm, with the approximate tumor age ranging from 18 to 21
days. In the case of spontaneous mammary tumors, age of
tumor refers to the time since the tumors were palpable. The
data given in Table 1 show that, regardless of the blood cell
type, there was no significant depression of catalase activity in
any of the cells of tumor-bearing mice as compared with
control (tumor-free) animals. The same also was true for liver
catalase activity.
To determine whether the phenomenon
of catalase
depression occurs in animals with large tumors, we made
activity measurements on blood and liver from C3H/HeJ mice
bearing spontaneous 20-mm mammary tumors; these tumors
were approximately 35 days old. The data in Table 2 show
again that there was no depression in the catalase activity of
livers of the tumor-bearing animals. However, total leukocytes
of spontaneous, mammary tumor-bearing mice did show
significant depression of catalase, compared with tumor-free
animals. Preliminary experiments indicated that lymphocytes
from these tumor-bearing animals also showed appreciable
depression of catalase activity. This finding is not surprising,
.since the lymphocytes make up approximately 80% of the
total blood leukocytes of these animals, as was determined by
differential counts of whole blood smears, with the use of
Wright's stain.
That leukocyte catalase was depressed while liver catalase
was unaffected is an interesting observation. To explain this,
one might conjecture that if the leukocyte surface membrane
of the tumor-bearing animal was sensitive to saponin, resulting
in partial leakage of catalase from the cell without apparent
change of gross morphological cell structure, this could
account for the low activity value for leukocyte catalase
observed in these animals. Table 2 also shows that, regardless
of whether livers were just homogenized or whether the
homogenates
were further treated sonically to release
additional catalase activity, there was no significant difference
in catalase activity when tumor-bearing
animals were
compared with their tumor-free counterparts. With regard to
erythrocyte catalase, the tumor-bearing mice actually showed
activity slightly higher than that of the controls.
Blood and liver catalase activity of mice bearing several
different kinds of transplanted tumors was measured, and the
data from these experiments are shown in Table 3. Liver and
leukocyte catalase activity of DBA/1J mice carrying 5-day-old
pleomorphic sarcoma (S-37) transplants, approximately 1.0
cm in size, showed only an insignificant level of decrease when
compared with tumor-free animals of the same strain, sex, and
age. A/J mice carrying 5-day-old transplants of a spindle-cell
sarcoma, Sal, approximately 1.5 cm in size, showed some
depression in leukocyte catalase activity, significant depression
in liver catalase, and no depression in erythrocyte activity.
Measurements also were made on blood and liver cell
catalase activity of 2-month-old C3H/HeJ mice bearing
6C3HED lymphosarcoma transplants, of 2 different sizes, 1.5
and 2.0 cm, corresponding to 5- and 10-day-old transplants,
respectively. While there was a modest depression in liver
Table 1
The catalase activity of blood and liver cells of 7- to 8-month-old C3H/HeJ mice
bearing spontaneous mammary tumors, 3 to 8 mm in size
Activity (SMCU°/1X 10" cells)
Total no. of mice
2830
20Total
Cell type
leukocytes
Erythrocyte
Livere16.1
Tumor-free
Tumor-bearing
±2.0d
2.8
31.8 ±2.2
38.3 ±2.4
78,163 ±8,45618.4± 71.825 + 3.259-14
% catalase depression0
16
80.6
Pc
>p >0.5
0.1 >p>0.05
0.6 >p >0.5
" SMCU, standard millicatalase activity unit (no. of nmoles of hydrogen peroxide decomposed per min per ml of reaction volume).
b % Catalase depression = ¡(activityof tumor-bearing animals)/(activity of tumor-free animals) X 100] - 100%.
c Determined by the t test with no assumption of equal variance (18).
d Mean ±S.E.
c Activity is expressed as SCU per mg liver protein. Measurements were made on sonically disrupted liver homogenates.
Table 2
The catalase activity of blood and liver cells of 7- to 8-month-old C3H/HeJ mice
bearing spontaneous mammary tumors. 20 mm in size
cells)Totalno.
Activity (SMCUa/l X 10"
mice29
of
typeTotal
3327
±2.0
±1.6
leukocytes
45.0 ±3.8
38.3 ±2.4
Erythrocyte
Liverc-d
72,718 ±11,939
78,163 ±8,456
Liverc'cTumor-free16.1 62,186 ±10,280Tumor-bearing9.4
55,157 ±13.522%
14Cell
catalase depression42
-18
7
11P0.05
>p
0.02 > p
0.8 >p
0.7 > p
> 0.02b
>0.01b
>0.7
>0.6
a SMCU, standard millicatalase activity unit (no. of nmoles of hydrogen peroxide decomposed per min per ml of reaction volume).
b Significantly different (p < 0.05) compared with control (tumor-free) animals.
c Activity expressed as SCU per mg liver protein.
d Measurements were made on sonically disrupted liver homogenates.
e Measurements were made on liver homogenates.
1192
CANCER RESEARCH VOL. 32
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Catalase Activity of Tumor-bearing Mice
Table 3
The catalase activity of blood and liver cells of 2-month-old mice bearing transplanted tumors
Activity (SMCU"/1 X 10" cells)
Strain
Transplanted
tumor
Cell type
Tumor-free
Tumor-bearing
leukocytesLiverb-cTotal
JA/JC3H/HeJC3H/HeJPleomorphicsarcoma
DBA/1
(S37)Spindle-cellsarcoma(Sal)Lymphosarcoma(6C3HED)eLymphosarcoma(6C3HED/Total
leukocytesLiver
b'cTotal
% catalase
depression
>0.40.4
>p
0.30.5
>p >
>0.40.01
>p
>0.001d0.1
>p
>p>0.050.7
>0.60.1
>p
>p>0.050.7
>0.60.6
>p
leukocytesErythrocyteLiverò'c29.033,73329.927,26718.626.950,75018.626.950.7503.62,9234.92,1402.71.93,6072.71.93.60724.
>0.5p
>p
>0.001d
" SMCU, standard millicatalase activity unit (no. of nmoles of hydrogen peroxide decomposed per min per ml of reaction volume).
b Activity expressed as SCU per mg liver protein. •¿
c Measurements were made on liver homogenates.
**Significantly different (p < 0.05) compared with control (tumor-free) animals.
e Lymphosarcomas (6C3HED) were 1.5 cm in size.
f Lymphosarcomas (6C3HED) were 2.0 cm in size.
leukocytesErythrocyteLiverb'cTotal
catalase
activity
of
animals
carrying
the
1.5-cm
lymphosarcoma transplant, the depression in liver catalase was
highly significant for animals carrying the 2.0-cm tumor. There
was no depression in erythrocyte catalase of animas carrying
lymphosarcomas of either size. Leukocyte catalase showed a
greater
depression
in animals
carrying
the smaller
lymphosarcoma, but because of the possibility that the cells of
this tumor can invade the blood vessels, it is possible that total
leukocyte catalase activity is a measure not only of the host
white cells but also of cells contributed by the invading
lymphosarcoma.
Indeed, differential
counts made with
Wright's stain on glass slide smears of whole blood from
animals carrying 2.0-cm lymphosarcomas showed that there
were on the average twice as many very large lymphoid-like
cells as there were normal blood lymphocytes. On the other
hand, these large lymphoid cells were not observed in blood
from tumor-free animals.
Chart 1 shows the correlation of the Beers and Sizer assay
with the Groves assay for liver catalase activity of both
tumor-free and tumor-bearing animals. It can be seen that over
the range of activities measured there is a general parallelism
between the values obtained by the 2 assay procedures, the
correlation coefficient being 0.94. Therefore, our results
would not have looked any different if we had used a
procedure for assaying for catalase plus peroxidase activities
which depends on measurement of the disappearance of
hydrogen peroxide (14, 16).
DISCUSSION
Of all the individual liver functions, one of the most marked
changes that occur in experimental animals bearing a wide
variety of tumors is the lowering of the catalase activity level
(11). The results of our experiments on liver catalase activity
of C3H/HeJ mice are in agreement with the classical studies of
Greenstein and Andervont (5, 6). From our experiments, we
conclude, as did they, that a significant level of systemic
catalase depression does not occur at the early stages of
tumorigenesis.
We have extended the studies of Greenstein by investigating
the effect of tumor size on leukocyte catalase activity of
C3H/HeJ mice carrying a spontaneous mammary tumor, as
well as the lymphosarcoma, 6C3HED. We also have looked at
other inbred mice strains carrying certain transplanted tumors.
Our data indicate that, as in liver, leukocyte catalase is not
depressed at the early stages of malignancy. When leukocyte
catalase did show depression, the phenomenon occurred when
the tumors had become appreciable in size, i.e., at least 1.5
cm. On the other hand, erythrocyte catalase levels were not
lowered, even in animals bearing large tumors. Conceivably,
the erythrocyte catalase may be insensitive to depression
because of a protective effect of hemoglobin, both proteins
having the same iron-protoporphyrin
IX group. Of course,
other explanations cannot be ruled out (13, 17).
Recently,
Higashi et al. (10), using immunological
techniques with anticatalase antibody, showed that depressed
liver catalase of tumor-bearing animals was due to both an
inhibition of enzyme activity (supernatant fraction) and an
actual decrease in absolute amount of enzyme (particulate
fractions). Whether this reduction in amount of catalase was
caused by a decrease in the rate of its synthesis or by
stimulation of its degradation was not examined. The
conditions of assaying for catalase made in our present study
obviously
cannot
distinguish
between
any of these
possibilities.
Although we were not able to see depression of liver
catalase activity of C3H/HeJ mice bearing large spontaneous
mammary tumors, we were able to show a significantly
marked decrease in liver catalase activity of the same strain of
mice when these animals carried large, transplanted
lymphosarcomas. Because the liver homogenates from both
sets of experiments were obtained by homogenizing liver
samples in buffer with a Potter-Elvehjem homogenizer, we feel
that this procedure is sufficient to demonstrate depressed liver
catalase activity, when indeed the phenomenon
exists.
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1193
Joel H. Kaplan and James N. Groves
Furthermore, even when we have homogenized the livers of
C3H/HeJ mice bearing spontaneous mammary tumors in large
volumes of cold distilled water, with a Waring Blendor, as
described by Nakahara (14), for release of total catalase
activity, we still have not been able to observe any significant
depression in liver catalase activity of these tumor-bearing
animals as compared with control (tumor-free) animals.
As a check to see that all leukocyte catalase activity from
C3H/HeJ mice was being measured, Triton X-100 was added
to a leukocyte cell suspension to 0.3% concentration. The
suspension was then homogenized by 20 strokes with a
Potter-Elvehjem tissue homogenizer. No change in catalase
activity could be observed, compared with catalase measured
from intact leukocytes. This was true regardless of whether
leukocytes were examined from tumor-free or from tumorbearing animals.
Whether depression of leukocyte catalase is indeed a true
systemic effect of tumorigenesis or whether it is actually a
reflection of a greater sensitivity of the leukocyte cell-surface
membrane of the tumor-bearing animal to saponin (used for
isolating leukocytes from whole blood) remains open to
question. In any event, regardless of mechanism, depression of
leukocyte catalase activity occurred only when the tumors had
reached a large size.
As in the case of mice (5), appreciable depression of hepatic
catalase activity has been observed in humans with advanced
cancers, while in individuals with regressing tumors there has
been a rise in activity (16). A parallel situation in regard to
leukocyte activity levels therefore might be likely, in which
case measurements of leukocyte catalase levels should have
prognostic value in the treatment of certain types of human
cancers.
ACKNOWLEDGMENTS
We are indebted to Mr. John L. Dorey and his staff at the Production
Department of The Jackson Laboratory for their technical expertise in
preparing the liver and whole-blood specimens used throughout the
course of this investigation. We also wish to thank Miss Marie Lynch
and Mr. Anthony Razzano for their expert technical assistance during
certain phases of this work.
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CANCER RESEARCH VOL. 32
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Liver and Blood Cell Catalase Activity of Tumor-bearing Mice
Joel H. Kaplan and James N. Groves
Cancer Res 1972;32:1190-1194.
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