Download Sequential Alterations in Mitochondrial Inner and Outer Membrane

[CANCER
RESEARCH
29, 1298—1306,
June
1969]
Sequential Alterations in Mitochondrial
Inner and Outer Membrane
Electron Transport and in Respiratory Control during Feeding of
Amino Azo Dyes ; Stability of Phosphorylation.
Correlation with
Swelling-Contraction
Changes and Tumorigenesis Threshold1
Joseph C. Arcos, Mattie J. Tison, Hans H. Gosch2, and Judith A. Fabian
Seamen's Memorial Research Laboratory, USPHS Hospital, New Orleans, Louisiana
University School ofMedicine,
New Orleans, Louisiana 70112
SUMMARY
In relation
to preceding
investigations
on the swelling
and contraction
properties
of rat liver mitochondria
during
the feeding of amino azo dyes, the respiratory
rate, the
P:O ratio, the respiratory
control
index, as well as the
NADH2 cytochrome
c reductase
and diaphorase
activities
were studied in rat liver mitochondria
during the time
course of feeding 0.06% 3'-methyl-4-dimethylaminoazobenzene
(3'-Me-DAB) and 2-methyl-4-dimethylaminoazobenzene
(2-Me
DAB), and in mitochondrma from 3'-Me-DAB-induced
hepa
toma. It was found that the P:O ratio remains at the
normal values throughout
16 weeks of feeding 3'-Me-DAB,
using pyruvate,
a-ketoglutarate,
or glutamate
as substrates;
the ratio is very slightly decreased throughout
the feeding
of 2-Me-DAB for 10 weeks. Mitochondria
from 3'-Me-DAB
induced
hepatomas
give a scattering
of P:O ratio values
with all three substrates indicating partial or total uncoupling
depending on the individual tumor.
Between 0 and 7 weeks (with a maximum at 3 weeks) the
Qo2 notably increasesduring feedingof 3'-Me-DAB;the in
crease is the largest with a-ketoglutarate,
less with glutamate,
and the smallest with pyruvate. This increase is due to tempo
rary release of the respiratory
control. Determination
of the
respiratory
control
indexes
showed,
in
fact,
considerable
minima at 3 weeks during feeding of 3'-Me-DAB, with either
of the three substrates or with 13-hydroxybutyrate.
The Qo2
values of tumor mitochondria
are lower than the Qo2 of nor
mal controls, and the decrease is statistically signfficant with
pyruvate and glutamate. Unlike 3'-Me-DAB, 2-Me-DAB causes
a large decrease of respiration with glutamate or pyruvate from
the very onset and throughout
the 10-week feeding period; in
contrast, with a-ketoglutarate
there is a gradual, moderate in
crease with a peak at 6 weeks.
Of the two outer membrane-localized
electron transport seg
ments, diaphorase activity shows a sharp minimum at 4 weeks
1Supported
by Research Grant CA-05431 from the National
Institute,
USPHS. Presented in part at the Ninth International
Congress,
Tokyo,
October
1966,
Abstract
Cancer
Cancer
S0318.
2Present address: Section of Neurosurgery,
Department
of Surgery,
University of Michigan Medical School, Ann Arbor, Michigan.
Received September 5, 1968; accepted January 30, 1969.
1298
701 18, and the Department
ofMedicine
(Biochemistry),
Tulane
during feeding of 3'-Me-DAB. This correlates with the minima
of swelling and “contraction―and with the onset of irrevers
ibiity of tumor induction with this dye. With NADH2 cyto
chrome c reductase the 4-week decrease is not seen; however,
immediately
following this period, from 5 weeks on, a tempo
nary, over fourfold, rise of activity is observed with a man
mum at 7 weeks. The high NADH2 cytochnome c reductase
activity at 7 weeks is largely observed in hepatoma
mito
chondnia. These changes of NADH2 oxidation
are either
absent or entirely different during administration
of 2-Me
DAB. The successive
changes observed
in mitochondnial
membrane-linked
functions probably represent steps of pro
gression in 3'-Me-DAB carcinogenesis.
INTRODUCTION
In the preceding report (9) alterations
of the swelling and
ATP-produced
“contraction―characteristics
of rat liver mito
chondria during the administration
of 3'-Me-DAB3 and 2-Me
DAB were described. There is a vast body of evidence indi
cating that swelling and “contraction―are in intimate relation
ship with the energy transducing
functions
of the mito
chondria
(33, 34, 52). Literally thousands
of reports have
appeared on the respiration,
phosphorylation,
and glycolysis
of tumors (as tissue slices and homogenates)
and of their
isolated mitochondria
in relation to the high glycolysis and the
postulated
“respiratory impairment― of the cancer cell (2—4,
13, 35, 46), and periodically cogent reviews have analyzed the
results of such studies (1, 27, 55, 57). However, while con
siderable effort has been devoted to ascertain the differences
and similarities of various tumors and tumor mitochondnia,
relative to normal tissues and their mitochondria,
investiga
tions are virtually absent in which respiration,
phosphoryla
tion, and other electron
transport-linked
processes
were
studied from the normal through the entire time-course of
progression
toward the malignant state. Yet such investiga
tions, by exploring successive stages of the premalignant
state,
3Abbreviations
used:
DAB,
4-dimethylaminoazobenzene;
DAB, 3'-methyl-4-dimethylaminoazobenzene;
dimethylaminoazobenzene
; Tris,
3'-Me
2-Me-DAB, 2-methyl-4-
tris-(hydroxymethyl)-aminomethane;
EDTA,
ethylenediaminetetraacetate
; NAD,
nicotinamide
adenine
dinucleotide; NADH2, reduced nicotinamide
adenine dinucleotide.
CANCER
RESEARCH
VOL.29
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Electron Transport and Respiratory Control in Carcinogenesis
Sprague-Dawley
male rats (Holtzman
Co., Madison, Wis.)
weighing 180—230 gm at the beginning of the experiments
were used. The general maintenance
and semisynthetic
diet of
the animals and the preparation
of liver and hepatoma tissues
before cell fractionation
were described previously (7, 9). The
mitochondria
used in all experiments,
except those in the
respiratory
control determinations,
were isolated in 0.44 M
sucrose in the presence of 0.001 M EDTA as described in an
respiratory
control determinations,
the flasks respiring in the
presence
of ATP “trap―
contained
in the side arm both
glucose and hexokinase at the same respective levels as speci
fled above; the flasks respiring in the absence of ATP
“trap―
contained only glucose in the side arm. The respiratory
control index represents the ratio: @il02 taken up in the pres
ence of phosphate acceptor over p1 02 taken up in the absence
of phosphate acceptor.
All reagents and the vessels were cooled in ice until the
actual incubations. The mitochondnia
were always introduced
last into the main compartment
of the vessels. In all experi
ments temperature
equilibration
was for 1 2 minutes. In the
measurements
of the P:O ratios the zero-time flasks were re
moved following equilibration
for the determination
of the
orthophosphate
levels present in the media before respiration.
Oxygen uptake was recorded at 5-mn intervals up to 30 min.
In the P:O ratio measurements
the remaining orthophosphate
was determined
in the respective flasks following respiration.
The respiratory control indexes presented in Chart 2 were cal
culated on the basis of the 30 min oxygen uptake.
For the determination
of the orthophosphate
levels for cal
earlier
culating
could yield valuable clues toward a better understanding
of the
neoplastic process.
This report describes a study of the Qo2 ‘
the P:O ratio, and
the respiratory control index of rat liver mitochondria
during
the time-course
of administration
of 3'-Me-DAB and 2-Me
DAB and in mitochondria
from 3'-Me-DAB-induced
hepa
toma. Moreover, the change in mitochondrial
diaphorase and
NADH2 cytochrome
C reductase
levels have been explored
during the feeding of the two dyes and in hepatoma
mito
chondria.
MATERIALS AND METhODS
report
(8).
The
mitochondria
used
for the
respiratory
control determinations
were isolated in 0.25 M sucrose follow
ing the procedure
of Holton et al. (26). The mitochondrial
contents of the final suspensions were determined
following
Lowry
@
et a!. (37)
for the
Qo2 , diaphorase,
and NADH2
cyto
chrome c reductase measurements,
and following Arcos et al.
(10) for the respiratory control determinations,
using a trichlo
roacetic acid-precipitated,
acetone-extracted,
and dried liver
mitochondrial
preparation as standard.
Respiratory
Rates, Respiratory
Control, and P:O Ratios.
Oxygen uptake
was measured in a conventional
Warburg
respirometer
at 30°C with air as gas phase. The center well
contained a strip of pleated filter paper and 0.2 ml 2 N KOH.
For the Qo2 determinations
in the oxidative phosphorylation
studies, and for measuring the inhibition of respiration by in
vitro added azo dyes, the following components
at the levels
indicated were added to the main compartments
of the reac
tion vessels: 40 jimoles, pH 7.4, Tris; 50 j.imoles, pH 7.4,
@2p@4; 20 j.zmoles KC1; 15 j@mo1es MgC12 ; 30 j.zmoles NaF;
0.5 @zmolesNAD; 0.03 .zmole cytochrome c; 6 i.@molesATP; 35
!lmoles a-ketoglutarate,
glutamate,
or pyruvate
(1 jimole
fumarate
was also included with pyruvate) ; mitochondria
equivalent
to the amount present in 500 mg normal liver
tissue; and 0.25 M sucrose to complete a final volume of 3.2
ml. In the in vitro studies with azo dyes the latter were intro
duced as 0.1 ml aliquots of methanol solutions and parallel
control experiments
with and without methanol were run. In
the side arm were placed 56 j@tmoles glucose and 2.0 mg yeast
hexokinase
(6 international
units). The pH of the medium,
monitored
in occasional flasks, after respiration was 7.0—7.2.
For the respiratory
control determinations
the incubation
medium was that of Holton et al. (26). A standard 6-mg mito
chondria was present in each flask. The essential difference
between the two respiratory media used in the present experi
ments is that the medium, adopted from Holton et at., also
contains a low level of EDTA and a close to fifteen times
higher level of KC1. The two respiratory
media were chosen
after systematic investigation of the optimal conditions. In the
the
P:O
ratios,
upon
removal
from
the
manometers,
the flasks were immediately
chilled in an ice bath and the
reaction stopped by addition of 0.2 ml ice-cold 5 N trichloro
acetic acid. The contents were adjusted to exactly 10 ml with
washings from the flasks and then centrifuged at 1000 X g for
15 minutes in the cold. Aliquots of the protein-free
super
natant
fluids
were
taken
up in standard
volumes
of 0.1 25 N,
pH 4.1, acetate buffer, and orthophosphate
was determined
following Lowry and Lopez (36).
In the Qo2 respiratory inhibition, and P:O ratio determina
tions, the controls are the averages of 1 1 experiments;
the
experimental
values are the mean values of 5 to 8 experiments.
All respiratory control indexes are the mean values of 5 exper
iments each.
NADH2 Oxidation.
Mitochondrial
diaphorase
activity was
measured (at 600 mz) in a medium of 0.02 M Tris buffer or of
0.02 M Tnis + 0.44 M sucrose (both at pH 7.4), following an
adaptation
of the procedure
of Edelhoch
et at. (18) and
Mahler
et a!. (38).
The
final
reaction
mixture
contained,
in a
total volume of 3.2 ml, 500 @igof NADH2, 0.12 jtmole
of 2,6-dichlorophenolindophenol,
and 1 .45 mg mitochondria
(whichcorrespondsto the amountpresentin 50 mg normal
liver tissue). The activity of the NADH2 cytochrome
c re
ductase was measured (at 340 m@) following an adaptation of
the procedure
of Zins et at. (60). The determinations
were
carried
out in a medium
of 0.05
M potassium
phosphate
buffer
or of 0.05 M potassium phosphate + 0.44 M sucrose, both at
pH 7.4. The final reaction
mixture contained,
in a total
volume of 3.2 ml, 170 pg of NADH2, 0.06 jimole of cyto
chrome C, and 2.3 mg mitochondria
(corresponding
to 80 mg
normal
liver
tissue).
In both
the
diaphorase
and
the
NADH2
cytochrome
C reductase
assays, absorbancy
changes were
recorded at 30 sec intervals at 20°Cin a Beckman DB spectro
photometer
fitted with a constant temperature
circulator. It
was always ascertained that the activity of the mitochondnia is
directly
proportional
mitochondrial
was adjusted
to their
concentration.
The
volume
of
stock suspension added in these determinations
so that the concentration
of the mitochondria
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in
1299
Joseph C. Arcos, Mattie J. Tison, Hans H. Gosch, and Judith A. Fabian
the linear segments of the rate curves by rheans of 2,6-di
chlorophenolindophenol
and NADH2 reference curves. In cx
pressing the cliaphorase activities in terms of NADH2 oxidized,
it was assumed that the reduction of the dye follows an exact
1: 1 stoichiometry.
The results on the two NADH2 oxidase
glutarate 4.27 (normal) and 4.93 (tumor), and with glutamate
3.85 (normal) and 5.57 (tumor). The mean Qo2 values of
hepatoma
mitochondria
are 7.43 with pyruvatc,
13.73 with
a-ketoglutarate,
and 21.99 with glutamate. These values are
lower than the respective normal liver rates (Charts la-c), and
the decrease is statistically
significant with pyruvate (P <
0.001) and with glutamate (0.05 > P > 0.02); with a-keto
activities
glutarate
the test
systems
was constant.
The levels of both
types
of
NADH2 oxidase activities were calculated from the slopes of
with
liver
mitochondria
are
the
averages
of 5 to
8
experiments;
with hepatoma mitochondria
the results are the
averages of 3 or 4 experiments.
Reagents. ATP (disodium salt, 99-100%),
NM), NADH2,
horse heart cytochrome
c (Type III), and 2,6-dichloro
phenolindophenol
(Grade I) were Sigma Chemical Company
products. Hexokinase
(lyophilized)
was from the Nutritional
Biochemicals Corporation
and the respiratory substrates from
the California Corp. for Biochemical Research. All other sub
stances were commercially available reagent grade products.
RESULTS
0.20
> P > 0.10.
Unlike 3'-Me-DAB,
feeding of the relatively
noncarcinogenic
2-Me-DAB does not produce the 3-week maximum ofthe Qo2.
On the contrary, the latter dye brings about a decrease of the
respiratory
rate from the very beginning and throughout
the
10-week period of administration
(Charts la and lc). This
depression
significant
of respiration
with
respect
with pyruvate
to 0 week
or glutamate,
which is
throughout
the whole
period (0.05 > P > 0.02 or better), is larger with glutamate
than with pyruvate. With a-ketoglutarate
(Chart lb) respira
tion is not depressed, but rather it increases to a small maxi
mum at 6 weeks and then returns to the normal level at 10
weeks (for this maximum 0.01 > P > 0.001).
Inner Membrane Electron Transport and Respiratory
Con
trol. Charts la—c present the Qo2 and P:O ratios of rat liver
mitochondria
(with pyruvate, a-ketoglutarate,
and glutamate)
during the time-course
of continuous
feeding of 3'-Me-DAB
The in vivo effects of the azo dyes on mitochondnial
respira
tion are not comparable to their in vitro effects. Chart 3 shows
that either DAB, 3'-Me-DAB, or 2-Me-DAB added in vitro in
and
strates. The inhibition,
which is concentration
dependent,
is
most pronounced with the noncarcinogen,
2-Me-DAB. These re
sults are in excellent agreement with the findings of McMurray
(39). However, Kielley (31) reported earlier that, in vitro,
3'-Me-DAB and o-aminoazotoluene,
but not DAB, inhibited
the respiration
of nondeficient
normal rat liver mitochondria
with glutamate, isocitrate, and succinate, but not with a-keto
2-Me-DAB
and
of
tumor
mitochondria
isolated
from
3'-Me-DAB-induced
hepatomas.
The charts show that, from
the onset of administration
of 3'-Me-DAB, the Qo2 increases
to reach a maximum at 3 weeks and then returns to or below
the normal level at 7 weeks. The 3-week maxima are statisti
cally significant; with all three substrates, P < 0.001 for the
populations
between 0 and 3 weeks and between 3 and 7
weeks. The magnitude of the maximum, in terms of percent
age increase relative to the control, increases in the order
pyruvate < glutamate < a-ketoglutarate.
Following 7 weeks,
the respiratory
rate returns to or somewhat above the control
level and the rate is maintained
to the end of the 16-week
feeding period. Chart 2 indicates that the 3-week maximum in
the Qo2 is due to the temporary
release of the respiratory
control. In fact, with either glutamate, a-ketoglutarate,
pyru
vate, or @3-hydroxybutyrate,
the respiratory
control index
@
variably
inhibits
mitochondrial
respiration
with
the
three
sub
glutarate and 13-hydroxybutyrate.
Phosphorylation.
Despite the dramatic
changes in mito
chondrial swelling and “contraction― (8, 9), Qo2, and respi
ratory control index, the phosphorylating
efficiency of liver
mitochondria
displays a remarkable constancy during the feed
ing of 3'-Me-DAB. Charts la-c show that the P:O ratios re
main essentially at the normal control level throughout
the 16
weeks
of dye
administration.
On the
other
hand,
the adminis
tration of 2-Me-DAB brings about slight but true decrease of
shows a deep minimum at 3 weeks during the feeding of 3'-Me
the P:O throughout
the 10 weeks (between 0 and 2 weeks and
DAB. The 3-week minimum is statistically
significant for all between 0 and 10 weeks P
0.05 or better). Mitochondnia
four substrates, with respect to both the 0- and 5- (or 10-)
isolated
from 3'-Me-DAB-induced
hepatomas
gave a large
week populations
(P < 0.001). Although the respiratory con
scattering of P:O ratios with all three substrates. Depending on
trol indexes given in Chart 2 were calculated from the 30 min the individual tumor preparations,
the mitochondria
are par
oxygen uptake values, it was noted that, in agreement with
tially or totally uncoupled.
It may be of significance that of
Aisenberg (3), the indexes are higher during the first 10—15 the three substrates used, uncoupling was the most severe with
miii of respiration
than during the subsequent periods. Thus,
pyruvate.
Oxidation of Extramitochondrial
NADH2. Study of the
the highest average respiratory control indexes of normal con
NADH2 cytochrome
C reductase and diaphorase activities, two
trol rat liver mitochondria,
calculated from the 10 or 15 min
oxygen uptake, were 7.7 with glutamate, 4.2 with a-ketogluta
outer membrane-localized
electron transport segments oxidiz
rate, 4.6 with pyruvate, and 5.9 with (3-hydroxybutyrate.
ing extramitochondrial
NADH2 (42, 50), revealed important
The Qo2 of hepatoma mitochondria
(Charts la-c) shows a
alterations
during administration
of 3'-Me-DAB. Although a
perceptibly greater scattering of values than the respiratory
relationship
between mitochondnial
diaphorase
and swelling
rates of liver mitochondria
from either normal or 3'-Me-DAB
(an innermembrane-linked
function)doesnot appearto have
been demonstrated,
Chart 4 shows a close parallel between
fed rats. This is seen in the greater standard deviation of the
mitochondrial
diaphorase activity and the previously observed
mean Qo2 with tumor mitochondria than with normal control
(8, 9) swelling changes during feeding 3'-Me-DAB. The appear
mitochondria;
thus,
the standard
deviation
with pyruvate
is
ance of the typical minimum at 4 weeks does not seem to be
1.11 for the normal and 2.51 for the tumor, with a-kcto
1300
CANCER
RESEARCH
VOL.29
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Electron Transport and Respfratory Control in Carcinogenesis
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25
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Weeksof feeding ow dyes
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Weeksof feedingazodyes
Charts la-c. Respiration and phosphorylation of rat liver mitochondria during the time-course of administration of amino azo dyes. The
carcinogenic 3 -methyl-4-dimethylaminoazobenzene
(3 -Me-DAB) and the relatively inactive 2-methyl-4-dimethylaminoazobenzene
(2-Me-DAB)
were fed at a 0.06 percent level in a semisynthetic diet. Individual values obtained with mitochondria from 3 -Me-DAB-induced hepatomas are
given. Mitochondria
were isolated
in 0.44 M sucrose containing
gsmoles, pH 7.4, tris-(hydroxymethyl)-aminomethane;
NAD ; 0.03
@zmo1ecytochrome
0.001 M EDTA. Each vessel contained
the following
basic reaction
mixture:
40
50 Mmoles, pH 7.4, KH2PO4; 20 @moles
KC1; 15 ismoles MgC12;30 ismoles NaP; 0.5 Mmole
c ; 6 @zmolesAlP ; 35 @smolessubstrate
(when pyruvate
was used, 1 iimole fumarate
was also present
as “sparker―);
10 mg glucose; 2 mg hexokinase; mitochondnia equivalent to the amount present in 500 mg normal liver tissue; and 0.25 M sucrose to complete to
a finalvolume of 3.2 ml. The center well contained a strip of pleated filter paper (‘-.-3.5
sq cm) and 0.2 ml 2 N KOH. Oxygen uptake was measured
(at 30 C) at 5-min intervals up to 30 min and expressed as zl 02 per mg trichioroacetic acid-precipitated, acetone-extracted, dry particulate
weight per hour. Orthophosphate was determined by the colorimetric method of Lowry and Lopez (36). The control values with normal
mitochondria
represent the mean values of 11 experiments;
the experimental
values, determined
feeding the dyes and with tumor mitochondria,
represent the mean values of 5 to 8 experiments.
@
with liver mitochondria
at different stages of
For the 3-week maximum in the Qo2@ with all
three substrates, the significance for true difference is P < 0.001 between the 0 and 3 week and between the 3 and 7 week populations. For the
decrease ofrespiration with glutamate or pynuvate during 2-Me-DAB feeding, 0.05 > P > 0.02 or better throughout the 10 weeks; for the 6-week
maximum with a-ketoglutarate 0.01 > P > 0.001 with respect to 0 and 10 weeks. The decrease ofrespiration oftumor mitochondria with respect
to the controls is significant with pyruvate (P < 0.001) and glutamate (0.05 > P > 0.02). For the small decrease of phosphorylation throughout
the feeding of 2-Me-DAB P 0.05 or better between 0 and 2 and between 0 and 10 weeks.
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1301
Joseph C. Arcos. Mattie J. Tison, Hans H. Gosch, and Judith A. Fabian
The pattern
of alterations
of NADH2 cytochrome
c re
ductase activity during the time-course of 3'-Me-DAB adminis
tration
.@
.——xglutamate
: a-ketoglutarate
—.@
pyruvate
is
entirely
different
(Chart
5)
than
that
found
for
diaphorase. The very small decrease seen here at 4 weeks is not
statistically
significant.
On the other hand, following
this
period, there is a sudden, over fourfold increase of activity in
the hypotonic
0.05 M phosphate medium, culminating
at 7
weeks; then, activity decreases again and approximates
the
normal at 12 weeks. The peak at 7 weeks is statistically signffi
cant with respect to both the normal control and the 12-week
populations
(P < 0.001). It is noteworthy
that this maximum
exactly coincides with the return of the Qo2 to or below the
normal level at the termination
of the period of respiratory
release (Charts la-c). Unlike the 4-week minimum in diapho
rase activity, the maximum in NADH2 cytochrome
c reductase
activity is considerably
influenced
by the tonicity
of the
medium. Chart 5 shows that in the sucrose-containing
(0.05 M
phosphate + 0.44 M sucrose) medium the gigantic peak almost
vanishes, which possibly indicates that the way by which
tonicity affects the activity of the NADH2 cytochrome
ductase
@-———@/3-hydroxybutyrate
may be more complex
than simple osmotic
c re
influence
on membrane permeability. The probability for true difference
for the small maximum
234567
at 9 weeks in the phosphate
+ sucrose
medium is 0.10 > P > 0.05.
Weeksof feeding3'-Me-DAB
Chart 2. Change in the respiratory control index of rat liver mito
chondnia during feeding of 0.06 percent 3'-methyl-4-dimethylaminoazo
benzene
(3'-Me-DAB)
in a semisynthetic
diet.
In these
experiments
the
-@-
isolation procedure of the mitochondria and the composition of the
respiratory
@
@
reaction
mixture
was patterned
after
Holton
et a! (26).
Mitochondria were isolated in 0.25 M sucrose. The exact mitochondrial
content of the fmal suspension was determined following Arcos et al.
(10) before the addition of mitochondria to the respiratory reaction
mixture. This reaction mixture had the following composition: 10
@zmoles,pH 7.4, tris-(hydroxymethyl)-aminomethane;
48 pmoles, pH
7.4, KH2PO4; 3 pmoles EDTA; 250 Mmoles KC1; 18 @smolesMgC12;36
@smolesNaF; 0.3 Mmole ATP; 25 @imolessubstrate (when pyruvate was
used, 1 @smolefumarate was also added as “sparker―)
; 10 mg glucose; 2
mg hexokinase only in the measurements of the respiratory rate in the
presence of ATP “trap―;
6 mg initochondria; 0.25 M sucrose to com
plete to a final volume of 3 ml. The center well contained a strip of
pleated filter paper (-‘.-3.5
sq cm) and 0.2 ml 2 N KOH. Oxygen uptake
was measured (at 30°C) at 5-min intervals up to 30 min and the respi
ratory control index was calculated on the basis of the terminal value.
Determinations of the respiratory rates with and without hexokinase
were carried out simultaneously. The respiratory control index repre
sents the ratio: oxygen uptake (Ml)with phosphate acceptor over the
oxygen
uptake
represent
the
cance
in the absence
mean
values
of phosphate
of 5 experiments
for the true difference
between
acceptor.
each.
These results
The statistical
the 0- and 3-week
signffi
values and
between the 3-week and 5 (or 10)-week values is P < 0.001.
k
a—Ketoglutorate
00
DAB
-.-
3-Me-DAB
-..
2-Me-DAB
Pyruvate
Glutamate
80
.@ .0
U
60
40
20
0
1002003004000
,,,,,_
1002003004000
Azo
00 200300
400
Dye
Chart 3. In vitro effect of amino azo dyes on the respiration of rat
liver mitochondria. Mitochondria were isolated in 0.44 M sucrose con
taming 0.001 M EDTA. Each vessel contained the following reaction
mixture: 40 @.&moles,
pH 7.4, tnis-(hydroxymethyl)-aminomethane;
50
@moles,pH 7.4,
@2@4 20 @molesKC1; 15 @smolesMgCl@;30@moles
NaF; 0.5
@zmoleNAD; 0.03
@smole cytochrome
c; 6 @imolesATP; 35
@smolessubstrate (when pyruvate was used 1 @tmolefumarate was also
added as “sparker―);
10 mg glucose; 2 mg hexokinase; a standard
amount
of mitochondria
corresponding
to that present in 500 mg fresh
liver tissue; the azo dyes 4-dimethylaminoazobenzene (DAB) and its
3'-methyl and 2-methyldenivatives (3'-Me-DAB and 2-Me-DAB) were
related to change of membrane permeability
since, except for
the magnitude of the rates, essentially identical curves were
obtained in either the hypotonic
(0.02 M Tris) or a sucrose
intioduced
in 0.1
ml methanol;
0.25
M sucrose
to complete
the reac
tion mixture to a final volume of 3.2 ml. The center well contained a
4-
strip of pleated filter paper (“-.‘3.5
sq cm) and 0.2 ml 2 N KOH. Oxygen
uptake was measured (at 30°C) at 5-min intervals up to 30 min and
expressed as @sl02 per mg trichloroacetic
acid-precipitated,
acetone
week minimum is statistically signfficant. The probabilities for
true difference between 0 and 4 weeks and between 4 and 12
weeks, in both the Tris and Tris + sucrose media, is P < 0.001.
extracted, dry particulate weight per hour. Control experiments were
carried out with and without methanol in the reaction vessel. These
results represent the mean values of 6 experiments each.
containing
1302
(0.02
M Tnis + 0.44
M sucrose)
medium.
The
CANCER
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Electron Transport and Respfratory Control in Carcinogenesis
occurs in rat liver mitochondria
3-Me-DAB
3'-Me-DAB.
in an early stage of feeding
Thus, while the respiratory
control
decreases
con
siderably between 0 and 5 weeks (Chart 2), which release
.8
expectedly
k
(Chart 1), the P:O ratio remains at the normal control level
throughout
the entire period of dye feeding (Chart 1). Since
this phenomenon
occurs with rat liver mitochondria
which
coincides
with
the
rise
of
the
respiratory
rate
normally exhibit high levels of respiratory control, a manda
tory conclusion is that in normal mitochondria the in vitro loss
I
of phosphorylating
ability is due to nonphysiologic
gross alter
ations of ultrastructure
(56) which are beyond the subtle
physiologic changes regulating the level of respiratory
control
in the cell. This suggests, in turn, that the level of respiratory
control
(i.e., the thrift
of substrate
utilization)
is regulated
by
Weeksof feeding
Chart 4. Change
during
the
in the diaphorase
time-course
activity
of feeding
methylaminoazobenzene
azobenzene
(2.Me-DAB)
of rat liver mitochondria
0.06
(3'-Me-DAB)
and
in a semisynthetic
percent
3'-methyl-4-di
2-Me-DAB
2-methyl-4-dimethylamino
diet. Diaphorase
activity
A ,,OO5MpO@
. mOO5MPO,n+O44MSucrose
was assayed by measuring the rate of decrease of absorbancy of 2,6dichlorophenolindophenol
at 600 mi.&.The tc@stsystem contained 500
i.ig NADH2, 0.12 Mmole 2,6-dichlonophenolindophenol,
and 1.45 mg
150
mitochondria, in a medium of 0.02 M, pH 7.4, tris-(hydroxymethyl)
aminomethane (Tris) in the absence or presence of 0.44 M sucrose
I00
(totalvolume3.2ml).Activities
werecalculated
fromtheratecurves
by means of a 2,6-dichlorophenolindophenol
reference curve and it was
50
assumed that the reduction of the dye by NADH2 follows an exact 1:1
stoichiometry.
The results with liver mitochondria
represent the mean
values of 7 or 8 experiments; the results with tumor mitochondria are
the mean values of 3 experiments. In the 3'-Me-DAB experiments the
statistical
significance
for the true differences
between
25
the 0- and 4-
week values and between the 4-week and 12-week values is P < 0.001 in
both media. In the 2-Me-DAB experiments in the Tris medium the dif
ferences between the activities are not significant through 8 weeks (0.20
> P > 0.10 or greater);
@
however,
for the difference
between
20
0 and 10
‘5
weeks 0.10 > P > 0.05. In the Tris + sucrose medium the small increase
between 0 and 3 weeks is significant, P 0.01.
@
@H@sornoO@
weeksof feeding
Administration
of 2-Me-DAB evokes smaller and entirely
different changes in the two NADH2 oxidase activities. In the
hypotonic
media the two oxidases showed closely resembling
patterns of alterations;
however, the one minimum and the
two maxima seen in the charts have unequivocal signfficance
only in the NADH2 cytochrome
c reductase
(Chart 5), for
which the probabilities
are 0.05 > P > 0.02 or better. In the
sucrose-containing
media the probabilities for the 3-week max
@
imum are P
0.01 for the diaphorase and 0.05 > P > 0.02 for
the NADH2 cytochrome
c reductase.
Release and P:O Ratio in Liver Mitochondria.
is commonly known that the loss of phosphorylating
mitochondria
originating
from
various
species
It
ability of
or tissues
coin
cides with the loss of respiratory control. Some instances are
known,
however,
I'2
Chart 5. Change in the NADH2 cytochrome c reductase activity of
rat liver mitochondria during the time-course of feeding 0.06 per
cent 3'-methyl4-dimethylaminoazobenzene
(3'-Me-DAB) and 2-methyl
4-dimethylaminoazobenzene
NADH2-cytochrome
(2-Me-DAB)
c reductase
activity
in
a
semisynthetic
diet.
was assayed by measuring
the
rate of the decrease of absorbancy of NADH2 at 340 mis. The test
system contained 170 @sg
NADH2, 0.06 @smolecytochrome c, and 2.3
mg mitochondria,
in a medium
of 0.05 M phosphate
buffer (pH 7.4) in
the absence or presence ox 0.44 M sucrose (total volume 3.2 ml). Activi
ties were calculated from the rate curves by means of a NADH2 refer
ence curve. The results with liver mitochondria represent the mean
values of 5 to 8 experiments; the results with tumor mitochondria are
the mean values of 4 experiments. In the 3'-Me-DAB experiments the
statistical significance for the true difference between the 0- and 7-week
and between the 7- and 12-week values is P < 0.001 in the phosphate
DISCUSSION
Respiratory
e
where
mitochondria
manifest
high phosphory
lation efficiency despite low levels or total lack -of respiratory
control (19, 25, 48; see also Ref. 34). A similar situation
medium. The apparent small minima at 4 weeks, in both media, are not
significant.
The small maximum
in the phosphate
+ sucrose medium
at
9 weeks, has 0.10 > P > 0.05 relative to the control. In the 2-Me-DAB
experiments
the comparatively
small variations
in the phosphate
mcdi
um are statistically significant: between 0 and 3, between 3 and 4, and
between
4 and
6 weeks
0.05
> P > 0.02
or better;
in the phosphate
+
sucrose medium the rise at 3 weeks, relative to 0 week, is significant,
0.05 > P > 0.02.
JUNE 1969
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1303
Joseph C. Arcos, Mattie J. Tison, Hans H. Gosch, andJudith
A. Fabian
mechanism(s)
independent
from the efficiency of phosphory
lation.
The remarkable
constancy of the P:O ratio appears to be
limited to certain substrates.
In the present study, the P:O
ratio was determined
with glutamate,
a-ketoglutarate,
and
pyruvate. However, Yamamoto et a!. (59) reported a decrease
of both the P:O ratio and the respiratory control index at 4
weeks during feeding of 0.06% 3'-Me-DAB, when succinate
was used as substrate
and ADP as phosphate
acceptor.
To ascertain the reality of this important substrate-dependent
difference, the experiments of Yamamoto et a!. will have to be
repeated under the present experimental
conditions.
The here-observed
release of the respiratory
control at 3
weeks immediately
follows or coincides with the maximum
level of 3'-Me-DAB binding in the liver, in whole homogenates
(40), microsomes
(5), and mitochondria
(8). This respiratory
release is a facet of the process of 3'-Me-DAB carcinogenesis,
since: (a) It approximately
coincides with the onset of irrevers
ibility in 3'-Me-DAB tumorigenesis
(8) and it immediately
precedes the deep 4-week minimum in the terminal extent of
swelling of liver mitochondria
during feeding of this dye (8, 9).
(b)
The
relatively
noncarcinogenic
2-Me-DAB,
which
reaches
the maximum level of binding in whole-liver homogenates
at
the 12th week of administration
(40), has a diametrically
opposite effect, Le., it inhibits respiration (with glutamate and
pyruvate) throughout
the 10-week feeding period. During the
feeding of 2-Me-DAB, in view of the large increase in the
amount of mitochondria
in the liver (8, 45, 51) also observed
in the present investigations,
the decreased respiration
with
giutamate and pyruvate may reflect the increased respiratory
control during intense anabolic activity (cf. 10, 11), possibly
restricted here to the mitochondria.
The way whereby 3'-Me-DAB induces the respiratory release
should now be briefly considered.
Bound 3'-Mc-DAB in the
mitochondria
(8) appears to be localized in the membraneous
constituents,
and the “structural protein― core of the inner
membrane repeating units may be the major site of binding
(9). Although little is known of the actual macromolecular
site(s) of respiratory control, it was established earlier that the
high energy state of whole mitochondria
(22, 23) and of mem
regarded as support for any specific hypothesis. Such decrease
may be the consequence
of factors other than the impairment
of electron transport.
For example, it appears that the in
hibitory effect of uncouplers of oxidative phosphorylation
on
mitochondrial
respiration may be due to the lack of availabil
ity of energy for penetration
(54). Conditions
of varying
osmolarity have been found to selectively affect the penetra
tion and oxidation
of certain substrates
(12, 28), and it is
possible that a similar situation prevails in the tumor mito
chondria
which
are resistant
to swelling and contraction
(7—9).
The present finding, that the oxidative phosphorylation
of
tumor mitochondria
was always uncoupled to some degree, is
in general agreement with the results of Devlin and Boxer (17)
on DAB-induced
hepatomas.
It may, of course, not be cx
duded that some of the loss of phosphorylating
ability may
have been due to the greater lability of hepatoma
mito
chondria than of liver mitochondria
under the standard condi
tions of isolation. Nevertheless, the extreme variations of the
efficiency of phosphorylation
with a given substrate in mito
chondria from individual tumor preparations appear to indi
cate the randomness of 3'-Mc-DAB-induced biochemical
lesions
and, hence,
lend circumstantial
support
to the thesis
(6) that chemicalcarcinogensare relativelynonspecificcell
poisons.
Extramitochondrial NADH2 Oxidation. Rat liver mito
chondria contain two diaphorases, the dicoumarol-sensitive
and rotenonc-insensitive
DT diaphorase which is equally active
with NADH2 and NADPH2, and a NADH2-specific
dicouma
rol-insensitive and rotenonc-sensitive
diaphorase which is prob
ably identical with the NADH2 dehydrogenase
of the main
respiratory chain (14). Another pathway of electron transport
from extramitochondrial
NADH2 is the NADH2 cytochromc c
reductase first described by Lchningcr (32) and clearly local
lied in the outer membrane (42, 50). However, reports from
several laboratories (16, 30) have raised the possibility that the
diaphorase and NADH2 cytochrome
c reductase activities arc
artifacts resulting from the transformation
of the NADH2 dc
hydrogenase
of the main
respiratory
chain.
brane fragments (41) is associated with a certain type of mito
chondrial morphology
or membrane-fragment
configuration.
Recent work indicates that the high-energy state is grounded
in an energized-twisted
configuration
of the inner membrane
Under the conditions of the present experiments,
the mito
chondrial suspensions were maintained in ice and used within
10 min after preparation.
Hence, comparison of the diaphorase
and NADH2 cytochrome
c reductase activities during feeding
of 3'-Me-DAB (Charts 4, 5) lends strong credence to the sepa
repeating units (24, 43), and the stability of this energized
rate identities
configuration
determinations
of diaphorase activity were carried out in the
absence of rotenone, it is likely, in view of the high rate of
electron transport in the main chain at 4 weeks (Chart 1), that
the 3'-Me-DAB curve in Chart 4 reflects exclusively the van
ations of the DPNH-activity of the DT diaphorase.
the
context
lytic
is the basis of the respiratory
of the
protein
proposal
that
control
“restructuring―
may take place in the basepieces
(29). In
of the
cata
of the mito
chondrial inner membrane in order to sterically accommodate
the dye-linked
modified
“structural protein― units
(9), the
temporary loss of the ability to take up the energized configu
ration may
rangement.
reflect
the
first
stage
of this
structural
rear
Qo2 andP:ORatioof TumorMitochondria.Althoughthe
controversy
about the group of phenomena called the “respi
ratory impairment
of the cancer cell― (58) appears still un
settled (compare Refs. 20 and 44), the writers feel that the
here-observed
decrease of the mean Qo2 of hepatoma mito
chondria (with pyruvate and glutamate) cannot at present be
1304
of these two NADH2 oxidases. Although
the
An intriguing finding is the close similarity between the
changes in the terminal extent of swelling (8, 9) and the
changes in diaphorase
activity (Chart 4) during feeding of
3'-Me-DAB.
Although
no relationship
between
the DT
diaphonase and mitochondrial
swelling has as yet been ne
ported, it is to be noted that dicoumarol, which is the most
potent inhibitor of this diaphorase (14, 15), also inhibits swell
ing (53). It should be recalled here that mitochondnial
swelling
does not occur under anaerobic conditions or when the respi
CANCER
RESEARCH
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Electron Transport and Respiratory Control in Carcinogenesis
ratory
chain
is blocked
by various
inhibitors;
however,
swelling
proceeds in the presence of an inhibitor if electron flow is estab
lished in only a segment of the chain by means of an artificial
electron acceptor above the block or an electron donor below
the block (reviewed in Ref. 33) . Moreover, the DT diaphorase
has been shown to link the oxidation of extramitochondnial
pyridinc nucleotides to the respiratory chain following the first
coupling site (1 5). For these reasons the close similarity be
tween the swelling and diaphorase changes during feeding of
3'-Mc-DAB suggests that the dye-induced
alterations
rcspon
sible for the 4-week swelling minimum involve also the cyto
chrome segment in the respiratory
chain and/or the external
diaphorase. This may provide an explanation for the finding of
Yamamoto
et al. with succinate (59), different from our re
sults with pyruvate, a-ketoglutarate,
and glutamate, since suc
cinate-initiated
electron
transport
also feeds into the main
chain following the first coupling site.
An alternate possibility is grounded in the observation that
substantial
quantities
of 2,6-dichlorophenolindophenol
are
bound to mitochondnia
by way of -SH groups, and this results
in the loss of 600 mj.@absorbancy analogous to the reduction
by diaphorase (21). Since some types of mitochondnial
swell
lag, and ATP-produccd
“contraction,―involve large changes in
the level of mitochondrial
-SH groups (47), it cannot be cx
cludcd
at present
that the apparent
sharp decrease
of
diaphorase may actually be the consequence
of the unavaila
biity of -SH groups at the critical 4-week period.
Schneider et at. (49) reported earlier the determination
of
NADH2-cytochrome
c reductase
activities
in liver mito
chondria isolated from rats fed 0.06% 3'-Me-DAB or 2-Me
DAB for 4 weeks. Their study, carried out with amounts of
mitochondria
present
in
100
mg liver
tissue,
yielded
results
indicating
an apparent
increase
of this enzyme
activity
following 2-Me-DAB administration.
However, these authors
have noted that if the increase of the number of mito
chondria
due to 2-Me-DAB administration
is taken into
account, enzyme activity per unit weight of mitochondnia
is
4. Aisenberg, A. C., and Morris, H. P. Energy Pathways of Hepatomas
H-35 and 7800. Cancer Res., 23: 566—568, 1963.
5. Arcos, J. C., and Arcos, M. Fine-Structural Alterations in Cell Par
tides
6. Arcos,
reductase
activity
of
mitochondnia
present
in
the
standard
amount of tissue; therefore, in this case, the enzyme activity
per unit weight of mitochondnia
remains practically constant.
Thus, their results are in excellent agreement with our findings
at 4 weeks (Chart 5).
I. Influence
of the Feeding
J. C., and Argus,
M. F. Molecular
Geometry
7. Arcos,
J. C., Gosch,
Alterations
in Cell
H. H., and Zickafoose,
Particles
During
and Carci
New Perspectives.
D. Fine Structural
Chemical
Carcinogenesis.
III.
Selective Action of Hepatic Carcinogens Other than 3'-Methyl-4Dimethylaminoazobenzene on Different Types of Mitochondnial
Swelling. Effect of Stimulated Liver Growth. J. Biophys. Biochem.
Cytol.,
10: 23—36, 1961.
8. Arcos, J. C., Griffith, G. W., and Cunningham, R. W. Fine Struc
tural
Alterations
II. Further
in Cell Particles
Evidence
for Their
During
Chemical
Involvement
Carcinogenesis.
in the Mechanism
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Carcinogenesis. The Swelling of Rat Liver Mitochondnia During
Feeding of Amino Azo Dyes. J. Biophys. Biochem. Cytol., 7:
49—60,1960.
9. Arcos, J. C., Mathison, J. B., Tison, M. J., and Mouledoux, A. M.
Effect of Feeding Amino Azo Dyes on Mitochondnial Swelling and
Contraction.
Kinetic Evidence for Deletion of Membrane
tony Sites. Cancer Res., 29: 1288—1297, 1969.
Regula
10. Arcos, J. C., Sohal, R. S., Sun, S. C., Argus, M. F., and Burch, G. E.
Changes in Ultrastructure and Respiratory Control in Mitochondnia
of Rat Heart Hypertrophied by Exercise. Exptl. Mol. Pathol., 8:
49—65, 1968.
11. Arcos, J. C., Stacey, R. E., Mathison, J. B., and Argus, M. F.
Kinetic Parameters
of Mitochondrial
Swelling. Effect of Animal
Age. Tissue Distribution
of the Mitochondrial
“Contractile Pro
tein.― Exptl. Cell Res., 48: 448—460, 1967.
12. Atsmon, A., and Davis, R. P. Mitochondrial
Respiration
Under
Conditions of Varying Osmolanity. Biochim. Biophys. Acta, 131:
221—233, 1967.
13. Burk, D., Woods, M., and Hunter, J. On the Significance
of
Glucolysis for Cancer Growth, with Special Reference to Morris
Rat Hepatomas. J. Natl. Cancer Inst., 38: 839—863,1967.
14. Conover, T. E., Danielson, L., and Ernster, L. DT Diaphorase. III.
Separation
of Mitochondrial
DT Diaphorase
and Respiratory
Chain. Biochim. Biophys. Acta, 67: 254—267,1963.
15.
Conover,
IV. Coupling
of
Extramitochondnial
Reduced
Pynidine Nucleotide
Oxidation
Mitochondnial
Respiratory
Chain. Biochim. Biophys. Acts,
268—280, 1963.
T. E., and
Ernster,
L. DT
Diaphorase.
to
67:
16. Cremona, T., Kearney, E. B., Villavicencio, M., and Singer, T. P.
Studies on the Respiratory
Transformation
Chain-Linked
of DPNH
Dehydrogenase
DPNH Dehydrogenase.
to
V.
DPNH.Cytochrome
Reductase and Diaphorase Under the Influence of Heat, Proteolytic
Enzymes, and Urea. Biochem. Z., 338: 407—422,1963.
17. Devlin, T. M., and Boxer, G. E. Comparison of the Metabolic Activ
ity of Normal Liver and Hepatomas
of the Rat. Intracellular
ACKNOWLEDGMENTS
Hydrogen
The authors are greatly indebted
to Dr. Mary F. Argus
Natarajan Venkatesan for helpful criticism of the manuscript.
Carcinogenesis.
nogenic Activity of Aromatic Compounds.
Advan. Cancer Res., 11: 305—471,1968.
actually slightly decreased. Similarly, they found that the de
crease in the number of mitochondnia
due to the feeding of
3'-Me-DAB accounts for the decrease of NADH2-cytochrome
c
During Chemical
of Aminoazo Dyes on the Swelling and Solubilization of Rat-Liver
Microsomes. Biochim. Biophys. Acts, 28: 9—20,1958.
and Dr.
18. Edelhoch,
Properties
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CANCER
RESEARCH
VOL.29
Downloaded from cancerres.aacrjournals.org on June 14, 2017. © 1969 American Association for Cancer Research.
Sequential Alterations in Mitochondrial Inner and Outer
Membrane Electron Transport and in Respiratory Control during
Feeding of Amino Azo Dyes; Stability of Phosphorylation.
Correlation with Swelling-Contraction Changes and
Tumorigenesis Threshold
Joseph C. Arcos, Mattie J. Tison, Hans H. Gosch, et al.
Cancer Res 1969;29:1298-1306.
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