Download Cytochrome Oxidase in the Liver of the Rat

Cytochrome
Oxidase in the Liver of the Rat Bearing
Walker Carcinoma 256*
ALICEANDREWSGREENEANDFRANCESL. HAVEN
(Department
of Biochemistry,
University
of Rochester School of Medicine and Dentistry,
The tumor-bearing host suffers systemic ef
fects which cannot be explained solely by the pres
sure, local invasion, or métastasesof the tumor.
One such systemic effect is the progressive loss of
carcass lipide during tumor growth which has been
demonstrated by Mider et al. (14, 15), by Haven
et al. (9, 10), and by Boyd et al. (2, 3) in rats bear
ing Walker carcinoma 256. Since lipides are known
to be associated with the cytochromes and may be
necessary for their enzymatic activity, the lipide
depletion in the tumor-bearer may affect the ac
tivity of the cytochrome system. Only two previ
ous reports on cytochrome oxidase in the tumorbearing host are available. Greenstein et al. (8)
found a normal level of cytochrome oxidase in
liver, spleen, and lymph node of two patients who
had died from leukemia. Both had received x-radiation prior to death. Using the livers of OsborneMendel rats bearing transplanted Hepatoma 31,
Shack (17) found no significant difference between
the cytochrome oxidase of the normal rats and
that of the tumor-bearers. The size of the tumors
and the number of animals, however, were not
stated. In order to obtain liver cytochrome oxidase
values for animals comparable to those in which
the lipide loss was observed, the present study was
made with rats bearing Walker carcinoma 256.
MATERIALS AND METHODS
Animals.—Male Rochester rats (Wistar-derived) were used except in the growth hormone
experiment, in which female Sprague-Dawley rats
were used. Young rats weighing between 100 and
150 gm. were given the Walker carcinoma 256 subcutaneously on the back by the trocar technic. The
animals were housed in groups of six, with free
Rochester, N.Y.)
access to water and Purina Fox Chow. Approxi
mately 4 weeks after transplantation, when the
tumor was 15 to 20 per cent of the body weight,
the animals were housed singly in basket cages
with wire mesh floors, and an individual record of
food consumption and body weight was kept.
In the growth hormone experiment, 26 female
Sprague-Dawley rats were observed until their body
weights reached a plateau. Thirteen of these rats
were then given daily intraperitoneal injections of
0.2 ml. of a solution containing 2 mg/ml of Somar1
(Armour somatotropin). The other rats received
daily injections of 0.2 ml. of sterile isotonic saline.
A total of thirteen doses was given to each animal
over a period of 14 days after which the animals
were sacrificed, liver homogenates prepared, and
cytochrome oxidase assayed as described above.
Diet supplements.—The diet supplements were
administered daily through a syringe fitted with a
round-ended needle. When the ball of the syringe
needle was placed well back on the rat's tongue
and the dose delivered quickly, the animal would
swallow reflexly. The supplement of the methyl
esters of the fatty acids of Wesson oil was prepared
by hydrolyzing the oil and esterifying the fatty
acids with methanol. The resulting mixture was
carefully neutralized. A daily dose containing 100120 mg. of methyl esters was administered to each
of six tumor-bearing and six normal rats for 7
days. The daily supplements of 80 mg. of methyl
linoleate2 and 0.05 mg. of pyridoxamine hydrochloride were administered separately to each of
six tumor-bearing and six normal rats for 5 days.
* This investigation was supported by Research Grant
C-669(C7) and Fellowship CF-5936 from the National Cancer
Institute of the Public Health Service. The material in this
paper was presented in part at the meetings of the Federation
of American Societies for Experimental Biology in Chicago,
April, 1957, and represents a portion of the thesis submitted
by the first author in partial fulfillment of the requirements for
the Ph.D. degree.
Received for publication February 1, 1957.
Preparations and assay.—Normal and tumor-bearing rats
were sacrificed by decapitation and drained of as much blood
as possible. The liver was quickly removed and the moist
weight obtained. A 5.0-gm. aliquot was taken from the liver
of normal animals. Since the liver of the tumor-bearing rat
contains more water than does the normal liver (1, 4, 12), the
aliquot of tissue in the case of the tumor-bearer was increased
to 5.5 gm. Each tissue sample was minced and placed in
separate ,'u-ml. portions of cold 0.44 Msucrose containing 0.45
1Courtesy of Dr. S. L. Steelman, Armour Laboratories,
Chicago, 111.
2Obtained from the Hormel Foundation.
613
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614
Cancer Research
ml. of 0.1 Mcitric acid. The samples were then homogenized in a
Dounce homogenizer (7) surrounded by ice, and the homogenates were diluted to 40 ml. with cold 0.44 M sucrose.
Aliquota from homogenates
prepared in this manner were
taken for the cytochrome oxidase assay. For the preparation
of mitochondria,
the procedure of Dounce, Witter, et al. (7)
was followed.
Dialyzed dry weight was obtained from duplicate 1-ml.
aliquota which had been dialyzed against five -.'-liter portions
of distilled water. Pat-free dry weight was obtained by ex
traction of dialyzed, lyophilized samples for 5 and 3 hours with
3:1 95 per cent ethanol-diethyl
ether, followed by two 3-hour
extractions
with 1:1 chloroform-methanol.
The remaining
material was then dried overnight in an evacuated desiccator
and weighed.
The cytochrome oxidase assay was carried out at 25°C.
by the manometric
procedure
described
by Stotz
prepared mitochondria,
and the mixture was incubated for 5
minutes at room temperature.
At the end of this time 0.65 ml.
of distilled water was added to the first set of flasks, and 1.0
ml. of 1.32 M sucrose and 0.1 ml. of 0.09 M versene were added
to the second set, thus making the final concentrations
of all
reagents in both sets of flasks the same. In the first set, how
ever, the mitochondria
were preserved as indicated by the
spectrophotometric
method of Cleland (5), whereas in the
second set of flasks the mitochondria
were swollen. After
the addition of all reagents the assay was carried out in the
usual manner.
RESULTS AND DISCUSSION
The cytochrome oxidase activity expressed
either per mg. of dialyzed dry weight or per total
liver was significantly greater in the tumor-bear-
(20). The
TABLE 1
CYTOCHROME
OXIDASE
ACTIVITY
OFLIVERHOMOGENATES
FROMNORMAL*
(N)
ANDTUMOR-BE
ARING
t (TB)RATS
TOTAL-BODT
TUMOR
PER CENT
WEIGHT
(gm.)
GBOOP
Mean
Standard deviation
Standard error of the mean
Significance of difference
* Twenty rats.
N
328
OF TOTAL
TB
332
BOOT WEIOHT
33.5
CTTOCHBOUE
OXIDASE
ACTIVITY
»iOjX 10-'/hr/
lil Oj/hr/mg
total liver
dry weight
N
TB
N
TB
283
452
112
136
±16
±20
±4
±4
P<0.01
±42
±80
±9
±15
P<0.01
t Twenty-eight rats.
TABLE 2
CYTOCHROME
OXIDASE
ACTIVITY
OFLIVERMITOCHONDRIA
FROMNORMAL
(N) AND
TUMOR-BEARING
(TB)LITTER-MATE
PAIRS*OFRATS
TOTAX. BOOT
WEIOHT
(gin.)
GROUP
Mean
Standard deviation
Standard error of the mean
Significance of difference
* Four pairs.
N
281
CYTOCHBOUE
OXIDASEACTIVITY
TUMOR
PEB
»il
Oz/hr/mg
dry weight
CENT
Or TOTAL
TB
346
BOOT WEIGHT
41.1
assay flasks contained 1.0 ml. of 0.1 Mphosphate buffer, pH 7.1 ;
0.8 ml. of 2 X 10~4 M cytochrome c (Sigma Chemical Company,
St. Louis); the tissue suspension; 0.3 ml. of 0.2 M hydroquinone
(Eastman Kodak Company, Rochester) in the side arm; and
sufficient distilled water to make a final volume of 3.0 ml. A
control flask contained the same constituents except that the
tissue suspension had been boiled for 10 minutes.
For the comparison of the cytochrome oxidase activity of
preserved and swollen mitochondria,
the assay was modified
as follows: Two sets of Warburg flasks were prepared, the
first for the assay of preserved mitochondria
and the second
for the assay of swollen mitochondria.
Into the first set were
pipetted 1.0 ml. of 0.1 u phosphate buffer, pH 7.1; 0.8 ml. of
2 X 10~* M cytochrome c; 1.0 ml. of 1.32 M sucrose; 0.1 ml.
of 0.09 M versene which had been neutralized to pH 7.1; and,
in the side arm, 0.3 ml. of 0.2 u hydroquinone.
Into the second
set of flasks were pipetted only the phosphate buffer, cyto
chrome c, and hydroquinone
(side arm) in the amounts indi
cated above, plus 0.65 ml. of distilled water. To all flasks was
then added 0.05 ml. (about 0.5 mg. dry weight) of freshly
N
TB
384
555
±66
±98
±33
±49
P<0.02
ill Oj/hr/mg fatfree dry weight
N
TB
475
742
± 86
±138
± 43
± 69
P<0.02
ers, as shown by the analyses of liver homogenates
from 20 normal and 28 tumor-bearing rats
(Table 1).
The liver mitochondria from four pairs of littermates, of which one partner of each pair carried a
tumor, were also assayed for cytochrome oxidase.
As shown in Table 2, the cytochrome oxidase ac
tivity for the mitochondria from the liver of the
tumor-bearer was significantly greater than that
of the normal, whether expressed on the basis of
dialyzed dry weight or of fat-free, dialyzed dry
weight.
When the individual values for cytochrome oxi
dase were compared with the size of the tumor, the
animals with the larger tumors were observed to
have the higher cytochrome oxidase levels. This
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GREENE ANDHAVEN—LiverCytochrome Oxidase in Tumor-bearing Rats
615
relation is shown in Chart 1. The increase became inal measurements were made gave the maximal
cytochrome oxidase activity for both the normal
demonstrable when the tumor weight was approxi
animal and the tumor-bearer. Therefore, the
mately 30 per cent of the total body weight.
greater cytochrome oxidase activity of the tumorThese findings have been examined to deter
mine whether the increase in cytochrome oxidase bearer cannot be explained by a greater permea
was truly a direct, or only an indirect, effect of the bility of the mitochondria to the substrate.
Since the uncoupling of oxidative phosphorylatumor on the host. The possibility that the deplet
ed and wasted state of the host might explain the tion is known to increase respiration (18), the in
findings was considered. The tumor-bearer may creased oxidase activity of the liver of the tumorhave less liver lipide or labile protein than the nor
bearer might represent a greater degree of un
mal animal.
coupling. To test this hypothesis, dinitrophenol
The decrease of these components in the tumor- was added to the assay system to assure complete
bearer would make the cytochrome oxidase activi
uncoupling. No increase in oxygen uptake was
ty based on dry weight appear greater than that observed as the result of dinitrophenol, with
of the normal animals. Since the increase was sig either preserved or swollen mitochondria of the
nificant both on dry weight and fat-free dry weight
bases, lipide differences could not explain the find
160
>ings. Similarly, the increase in cytochrome oxidase
H
concentration could not have been due to the loss >
of other liver protein from inanition of the host.
Wainio (23) has shown that although rats suffering
from protein depletion have a higher cytochrome
oxidase concentration in the liver, these animals
o i 120
have smaller livers and consequently less total
cytochrome oxidase per liver than do normal ani
o o
mals. The tumor-bearing rat, on the other hand,
UJÃŽ*
100
has a higher total cytochrome oxidase per liver
owing to an enlarged liver as well as an increased
concentration of the oxidase. Therefore, differ
ences in liver protein would not explain the ele
IO
20
30
40
50
vated cytochrome oxidase activity of the tumorTUMOR
SIZE
bearer.
% OF TOTAL BODY WEIGHT
Another possible explanation for the increased
CHART1.—Relation
between relative cytochrome oxidase
cytochrome oxidase was considered. If the mito
activity
and
tumor
size.
chondria from the liver of the tumor-bearing rat
were more permeable to the hydroquinone sub
strate than the mitochondria from the normal rat, normal or tumor-bearing rat. From these findings,
the conclusion may be drawn that phosphorylathen the greater access of the enzyme to the sub
strate might be reflected in the cytochrome oxidase tion in the mitochondria of both the normal liver
activity. Such an explanation did not appear very and the liver of the tumor-bearing rat was com
likely because the substrate was present in excess pletely uncoupled by the time of assay. The in
and the assays were performed in the presence of crease in cytochrome oxidase observed in the tu
phosphate, which is known to cause a swelling and mor-bearer cannot, therefore, be due to a greater
disruption of the mitochondria. The possibility uncoupling in the mitochondria of the tumorwas tested, nevertheless, by assaying both homog- bearer as compared with the normal.
enates and mitochondria for cytochrome oxidase
Still another possible explanation for the results
was examined. Kunkel and Williams (11) and Tulunder two sets of conditions, one of which pre
served the mitochondria and the other of which pule and Williams (22) have reported an increase
caused them to swell. The procedure is described in cytochrome oxidase in rats suffering from essen
under "Materials and Methods."
tial fatty acid deficiency. The fox chow diet, which
The oxygen uptake for the swollen mitochon
the animals in our experiments received, contains
dria was approximately double that of the pre
an adequate supply of fatty acids, both essential
served mitochondria. No difference, however, was and nonessential, for the growth and maintenance
of normal rats. The tumor-bearer, however, may
found between the behavior of the mitochondria
from the normal animal and those from the tumor- suffer a fatty acid deficiency in spite of an appar
bearer. The assay conditions under which the orig
ently adequate diet because the tumor causes a
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616
Cancer Research
mal animal and the tumor-bearer.
Finally, an attempt was made to explain the
increase in cytochrome oxidase as an adaptive re
sponse to tumor growth and consequent rapid pro
tein synthesis. For example, during lactation,
when accelerated protein synthesis is occurring,
the mammary gland has a much higher cyto
chrome oxidase activity (16, 21). To test the hy
pothesis that the demands of progressive tumor
growth caused the increase in cytochrome oxidase,
an experiment was designed to subject normal rats
to a similar stress of rapid growth by the use of
growth hormone. No previous reports on the effect
of growth hormone on liver cytochrome oxidase of
intact rats have been found. The animals were
depletion of the fat stores (9) and because rapidly
growing tissue requires essential fatty acids (6,
19). Thus the increase in cytochrome oxidase in the
liver of the tumor-bearing rats might be caused by
an essential fatty acid deficiency arising from the
depleting effect of the tumor on the host.
Kunkel and Williams (11) were able to reverse
the change in cytochrome oxidase and to lower it to
normal levels by adding 5 percentcorn oilor 100mg.
of methyl linoleate to the daily ration. Tulpule and
Williams (22) likewise prevented the cytochrome
oxidase increase by including methyl linoleate and
vitamin B6 in the basal diet. On the basis of this
evidence, experiments were carried out in which
the diets of the tumor-bearing rats and their nor
TABLE S
EFFECTOFDIET ONCYTOCHROME
OXIDASEACTIVITYIN NORMAL(N)
ANDTUMOR-BEARING
(TB) RATS*
FOXenow +ME-EBTEHS
or WESSONOIL
Fox CHOW
GROUP
Mean cytochrome oxidase activity
(M)Oj/hr/mg)
Significance of differences
N
95
FATTY ACIDS
TB
113
N
107
P<0.05
P<0.01
P<0.1;
>0.05
TB
ISl
Fox CHOW,MELINOLEATE, AND
VITAMIN Bl
Fox CHOW
N
101
TB
146
TB
194
N
US
P<0.01
P<0.01
P<0.02
* Sii rats per group.
TABLE 4
EFFECTOFGROWTHHORMONEONCYTO
CHROMEOXIDASEACTIVITY
Saline-treated
No. animals
Av. weight gain
(gm/14 days)
Av. food consumption
(gm/day)
Av. cytochrome oxidase
(M!0,/hr/mg)
* Standard error of the mean.
13
10
Growth hor
mone-treated
13
29
17
18
103+ 6*
102±6*
mal controls were supplemented with the methyl
esters of Wesson (cottonseed) oil fatty acids or
with methyl linoleate and vitamin Be in order to
evaluate the possibility of a dietary fatty acid
deficiency.
As shown in Table 3, the effect of the methyl
esters of the Wesson oil fatty acids is not signifi
cant by the usual standard of P < 0.05, but the
methyl linoleate and vitamin B6 supplements have
increased the difference between the normal and
the tumor-bearer significantly (P < 0.02). From
this the conclusion may be drawn that the increase
in cytochrome oxidase found in the tumor-bearer
was not due to an essential fatty acid deficiency of
the type described by Williams. It appears, in fact,
that the dietary supplement has exaggerated rath
er than eliminated the difference between the nor-
treated as described under "Materials and Meth
ods." The results are shown in Table 4. There was
no increase in the activity of cytochrome oxidase
as the result of the treatment with growth
hormone.
In conclusion, no explanation has been found
for the increase of cytochrome oxidase in the liver
of the tumor-bearing rat. The possibility remains
that this increase represents the adaptation of the
host to tumor growth. Since the tumor-bearing rat
has demonstrated an increased caloric requirement
and expenditure (13,14), the elevated cytochrome
oxidase may be part of a general increase in oxida
tion. The report that some cancer patients have an
increased basal metabolic rate (24) is in accord
ance with this hypothesis. Such a general increase
in oxidation would partly explain the rapid deple
tion of the host's lipide stores. Further work must
be done to determine how the increase in cyto
chrome oxidase is related to metabolic changes in
the tumor-bearing rat.
SUMMARY
1. Liver homogenates from tumor-bearing rats
carrying the Walker carcinoma 256 had greater
cytochrome oxidase activity than homogenates
from normal rats, whether the activity was ex
pressed on the basis of dialyzed dry weight or of
total liver.
2. Isolated mitochondria from the liver of tu-
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GREENE ANDHAVEN—LiverCytochrome Oxidase in Tumor-bearing Rats
mor-bearing rats also had an elevated cytochrome
oxidase activity expressed on the basis of dialyzed
dry
weight
or of fat-free,
diulyzcd
dry
weight.
3. The cytochrome oxidase activity increased
with the size of the tumor, the higher cytochrome
oxidase levels being found in animals with larger
tumors. The increase in oxidase activity became
demonstrable when the tumor weight reached 30
per cent of the total body weight.
4. The increase could not be explained by a
greater mitochondrial permeability to substrate
on the part of the tumor-bearer.
5. Similarly, the increase could not be explained
by a greater degree of uncoupling of oxidative
phosphorylation by the tumor-bearer.
6. Although essential fatty acid deficiency may
cause an increase in cytochrome oxidase activity,
it was not the explanation for the increase in the
tumor-bearing rat. Dietary supplements of the
methyl esters of Wesson oil fatty acids or of meth
yl linoleate and vitamin B6 enhanced rather than
prevented the increase in cytochrome oxidase in
the liver of the tumor-bearer.
7. Treatment of normal animals with 0.4 mg. of
growth hormone per day for 13 days did not affect
the activity of the liver cytochrome oxidase.
8. The possibility that the increased cyto
chrome oxidase may represent the response of the
host to the demands of the tumor for increased
oxidation is discussed.
ADDENDUM
After this manuscript was prepared, Wood et al. reported an
increase in the activity of the tryptophan peroxidase-oxidase
(TPO) system in the liver of mice bearing the transplan table tu
mor, Lewis sarcoma 241 (Cancer Research, 16:1053-58, 1956).
The increased cytochrome oxidase activity which we have found
was not due to TPO since (a) TPO is a soluble enzyme, present
for the most part in the supernatant fraction, while our in
creased cytochrome oxidase activity was observed in the
mitochondria as well as the homogenate; and (6) the maximum
oxygen uptake calculated from the data of Wood et al. is less
than 1 ¡A.of oxygen/hr/mg dry weight, while the oxygen
consumption of the liver preparations of our tumor-bearers ex
ceeded the normal by 30 /J. of oxygen/hr/mg dry weight. The
similarity in response of the two enzyme systems to progressive
tumor growth is interesting and undoubtedly of importance in
the tumor-host relationship.
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Cytochrome Oxidase in the Liver of the Rat Bearing Walker
Carcinoma 256
Alice Andrews Greene and Frances L. Haven
Cancer Res 1957;17:613-617.
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