Download Biochemical and Quantitative Histochemical

[CANCER RESEARCH 43, 4850-4855,
October 1983]
Biochemical and Quantitative Histochemical Study of Reduced Pyridine
Nucleotide Dehydrogenation by Human Colonie Carcinomas
Norberto A. Schor1 and Cornells J. Cornelisse
Department of Pathology,
Netherlands [C. J. C. I
Tulane University School of Medicine, New Orleans, Louisiana 70112 [N. A. S.], and Department of Pathology, Leiden University
ABSTRACT
This study shows a marked increase in the activity of the
soluble enzyme DT-diaphorase and of the histochemical activity
of the reduced nicotinamide adenine dinucleotide and reduced
nicotinamide adenine dinucleotide phosphate nitroblue tetrazolium menadione-mediated reductases in human colonie carcino
mas when compared with the enzymatic activities of portions of
the colon uninvolved by the carcinomatous process. The activity
of the reductases in histological sections was quantitated with a
microphotometer. It is believed that the increase in histochemical
nitroblue-tetrazolium reductase activity in the histochemical re
actions in colonie carcinomas is a real reflection of the activity of
the DT-diaphorase, because the increase in the dehydrogenation
of reduced nicotinamide adenine dinucleotide equals the dehy
drogenation of reduced nicotinamide adenine dinucleotide phos
phate when measured biochemically in the soluble fraction, or
histochemically, by microspectophotometry
in tissue sections;
meanwhile, the biochemical dehydrogenation of NAD(P)H by the
paniculate fractions shows that the enzymatic activities are not
altered by the neoplastic process. The biological significance of
these changes is discussed in the text.
INTRODUCTION
Since the work of Wattenberg in 1959 (30), it has been known
that human colonie carcinomas show an increase in the histo
chemical activity of enzymes NAD and NADP diaphorase. Those
findings have been confirmed by other workers (12, 17). The
increase in enzymatic activity is not a uniform change. The
majority of tumors shows an increased activity when compared
with the normal colonie mucosa, but others show either the same
or decreased activity.
The reduction of a tetrazolium salt in tissue sections when
NADPH or NADH is used is not due to a single specific enzyme.
Any of the different reduced pyridine nucleotide dehydrogenases
localized in the mitochondria, microsomes, or supernatant of the
cell may be responsible for this effect.
The main purpose of the present work was the study of the
different NAD(P)H-dehydrogenating enzymes of colonie carcino
mas. We tried to determine which enzymes (if any) were affected
by the neoplastic process and to observe if a quantitative cor
relation could be established between the biochemical and the
histochemical changes.
The enzymes studied by biochemical methods in this report
were the DT-diaphorase and the mitochondrial and microsomal
NADH and NADPH-cytochrome c reductases and the NADHand NADPH-dichloro-phenolindophenol
reductases. The histo
chemical activity of the diaphorases was assayed using reduced
1To whom requests for reprints should be addressed.
Received January 13,1983; accepted June 28, 1983.
4850
The
pyridine nucleotide, NBT2 and vitamin K3. These enzymes were
studied following a technique developed by Hack and Helmy (8),
which uses a brief fixation by formalin vapors to avoid the
diffusion of the reaction products in the incubation media. The
histochemical products were quantitated by scanning histophotometry. A positive correlation has already been observed be
tween the increase in activity of the DT-diaphorase and the
NAD(P)H-NBT reductase in the lymphatic system of the rat after
the administration of polycyclic hydrocarbons (19, 23). In mouse
skin, there is also a correlation between the increase in tetrazo
lium salt reduction and the increase in DT-diaphorase activity
after carcinogenic applications (11). Furthermore, the activity of
the DT-diaphorase is increased in some experimental tumors
(20-22).
MATERIALS AND METHODS
The tumors and portions of the normal mucosa of the uninvolved
colon were obtained immediately after surgery in the pathology depart
ments of different New Orleans hospitals. The tissues were frozen
immediately and kept at -25° until the time of processing.
In Table 1, the main clinical (age and sex) and anatomical features
(localization and degree of invasion) of the tumors used in this study are
enumerated.
For the biochemical determinations, 10 g per 100 ml homogenates
were prepared in 0.1 M phosphate buffer (pH 7.4) with 0.154 M potassium
chloride. The homogenates were then subjected to differential centrifugation. The first step was a sedimentation at 2000 rpm for 5 min to
discard nuclei and large cellular debris. The tissues were then transferred
to an ultracentrifuge where 2 fractions were obtained; one after 20 min
at 10,000 x g and the other after 90 min at 100,000 x g, thus providing
a mitochondrial, a microsomal, and a soluble fraction.
The following biochemical techniques were followed for the enzymatic
determinations. All of the concentrations listed are the final concentra
tions present in the incubation media: (a) NAD(P)H diaphorase: DCPIP,
0.048 DIM; phosphate buffer (pH 7.4), 0.045 M; and NAD(P)H, 0.33 mw.
The reaction was initiated by NAD(P)H and followed for 1 min at 600 nm.
The extinction coefficient of 22.1 HIM was followed for the calculations;
(fa) NAD(P)H cytochrome c reductases: cytochrome c, 0.018 mM;
NAD(P)H, 0.46 mw; phosphate buffer (pH 7.4), 0.035 M; and potassium
cyanide, 1.1 mw. The reaction was started with NAD(P)H and followed
for 1 min at 550 nm. The molar extinction coefficient of 19.3 mM for
cytochrome c was used for the calculations; and (c) malic and lactic
dehydrogenases: the same incubation medium was used [Tris buffer (pH
7.4), 0.021 M; NADH, 0.14 mM], and the substrates were oxaloacetic
acid, 1.4 mM [for the malic dehydrogenase) and pyruvate, 1.4 mM [for
the lactic dehydrogenase]. The oxidation of NADH was followed at 340
nm for 1 min. The 6.22 mw extinction coefficient of NADH was used for
the quantification of these enzymes. Proteins were determined by a
micromodification of Lowry's method (7).
The fragments of tissue used for histochemical purpose were frozen
again on a formulation of water-soluble glycols and resins over a mixture
2 The abbreviations used are: NBT, nitroblue tetrazolium; DCPIP, dichlorophenolindophenol.
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Human Colon Cancer Enzymology
Table 1
Main clinical and anatomical features ol tumors used
absorbances, the frequency distribution of the local absorbance values
and the mean absorbance value were recorded. The use of microphotometry
PatientN.
for the evaluation
of enzyme activity
using tetrazolium
salt
classificationBACCCCCCBCCCCCCACCCCCCCCBCCCCC
reduction on tissue sections has been reported (1, 26).
F.J.
D.P.M.E.
E.
RESULTS
F.G.
S.W.
Biochemical Determinations. The results obtained with the
W.L.A.
enzymes of the soluble fraction are shown in Table 2. Both the
flexureCecumSigmoidSigmoidCecumRectumCecumCecumRectumCecumSigmoidSigmoidRectumDescendingSigmoidTransverseRectumTransverseSig
D.W.
NADH and NADPH-DCPIP reductases are increased 3-fold in the
B.V.
P.D.
E.
tumors when compared with the normal mucosa. The DCPIP
M.H.
R.
reductases in the soluble fraction are an indication of the DTG.E.
diaphorase activity (4). The increase of the diaphorase activity is
G.V.
P.
W.LW.
statistically significant (p < 0.001). The increase in lactic and
D.M.
B.
malic dehydrogenase confirms results already published (25).
J.R.
W.R.
W.
These results show that the magnitude of the increase is larger
D.D.
E.
for the diaphorase (200%) than for the lactic and malic dehydroB.P.C.
genases (35 and 55%, respectively).
S.C.
C.
A.N.
We also have determined the activity of the NADPH and NADH
A.F.l.
dehydrogenases in the mitochondrial and microsomal fractions.
S.W.
G.
We used 2 different acceptors, DCPIP and cytochrome c. When
T.E.
B.L.W.
H.
DCPIP was used, no differences were observed between the
R.C.
tumors and the mucosa (Table 3), but when cytochrome c was
M.H.
H.
Z.J.J.
used as an acceptor, there was a small increase, not statistically
flexureSigmoidTransverseDuke's
R.T.
L.
significant, in the activity of the NADH-cytochrome c reducÃ-ase
D.M.
D.
of the mitochondrial fraction (Table 4). In the particulate fractions,
P. L.SexMMMMFMFMMFFMMFFFMFMFMFMMMMMFMMAge(yr)856775616687758176577255625546645964565463676067515874776567LocationSigmoidSigmoidSigmoidSigmoidSigmoidRec
of dry ice and acetone. They were cut ¡na cryostat at 7 pm. The sections
were briefly fixed in formalin vapors (between 1 and 2 min) and then
stained with the following technique: phosphate buffer (pH 7.4), 0.062
M; NBT, 0.48 mw; NAD(P)H, 0.042 mw; and vitamin K, 4.2 mw. After the
staining, the sections were washed in water, dehydrated in alcohol,
cleared in xylene, and mounted with permount.
Histophotometry. The amount of histochemical reaction product was
measured by histophotometry
on a Leitz MPV II microphotometer
equipped with a 0.5-nm Zeiss scanning stage. Scanning absorbance
measurements were done with green light from a stabilized halogen lamp
using a 558-nm narrow-band interference filter.
A x25 oil immersion objective was used with a x50 condenser
objective. The field diaphragm was 2 mm, and a 0.2-mm measuring spot
was used. The actual size of the measuring spot in the image plane was
2 ¿im.The stage movement and the signal processing were controlled
by the HYDACSYS (29) software package running on a PFP 11/10
minicomputer (Digital Equipment Corporation, Maynard, Mass.). Using a
step size of 10 /¿m,rectangular fields were scanned yielding an average
of 700 measuring points/field. Care was taken to include in each field an
unstained area, usually the central part of a gland, to serve as a
background for calculation of the 100% transmission intensity. This value
was automatically computed from the peak given by the background in
the histogram of the measured light intensities displayed on a Tektronix
4010 graphic terminal by the HYDACSYS program.
Four histological sections were analyzed quantitatively by the microphotometer, with 2 using NADH as a substrate and the others using
NADPH as a substrate. In each section, 7 measurements were made.
Each measurement was the equivalent of one gland. The sections
measured were consecutive sections, since the main purpose of these
measurements was to establish whether the microphotometer could
discriminate between differences of stainings of the NADH and the
NADPH-K3 reducÃ-ase. The same glands were measured in each section,
and the glands were located in the sections with the help of the scanning
stage.
In cases where no proper peak of background values was discernible,
the 100% value of the transmission was set with aid of a cursor at the
right border of the histogram. After conversion of the transmissions into
there was always a greater rate of reduction when NADH was
used as the substrate. When DCPIP was used as an acceptor,
the difference between the rates of NADH and NADPH dehydrogenation was not as remarkable as when cytochrome c was
enzymesMucosa
Table 2
Soluble
DCPIP"19±1.8C DCPIP323.5
± 2.3
TumorNADH- 55 ±8.1NADPH- 75.4 ±10.6Malic
dehydrogenase"572
dehydro
genase6487
±56.4
±52.3
769 ±54.7Lactic 756 ±54.7
pmol of reduced DCPIP per ^g protein present in the incubation media per min
for 30 samples.
" pmol of oxidized NADH per ^g protein present in the incubation media per min
for 30 samples.
c Mean ±S.E.
Table 3
Particulate enzymes, DCPIP reduction
Mitochondrial3Mucosa
DCPIP53.0
±3.9*
DCPIP22.6
DCPIP31
DCPIP18.5
±13.2
.6 ±2.5
±2.0
TumorNADH- 52.3 ±9.5NADPH-15.2± 7.5Microsomal3NADH32.3 ±3.0NADPH-15.8 ±1.9
* pmol of reduced DCPIP per ^g protein present in the incubation media for 30
samples.
" Mean ±S.E.
Table 4
Particulate enzymes, cytochrome c reduction
Mitochondrial3Mucosa
c72.9±5.36(24)c
chrome
c13.1
chrome
cyto
c49.5
chrome
c8.1
chrome
±1.8(24)
±4.4 (27)
±0.8 (27)
47.1 ±4.5 (27)NADPH-cyto8.3 ±1.1 (27)
86.0±7.7 (24)NADPH-cyto11.2 ±1.5 (24)Microsomal3NADH
TumorNADH-cyto
a pmol of reduced cytochrome c per ^g present in the incubation media per min.
6 Numbers in parentheses, number of samples analyzed.
c Mean ±S.E.
OCTOBER 1983
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4851
N. A. Schor and C. J. Cornelisse
used as an acceptor. This effect is probably due to the presence
of diaphorase activity in the paniculate fractions, because it is
difficult to clean the enzyme from mitochondria and microsomes,
and the presence of diaphorase does not affect the reduction of
cytochrome c, because the DT-diaphorase does not reduce
cytochrome c if menadione is not present in the incubation media
(4)Chart 1 shows the correlation between the biochemical reduc
tion of DCPIP by NADPH and NADH in the soluble fraction. The
value of the correlation coefficient is 0.94. Chart 1 shows that
some tumors have low activity, but the majority of the tumors
show a high activity. More important is the correlation between
the values obtained with NADH and NADPH.
Histochemical Results. Figs. 1 to 3 show the histochemical
staining for the NADH-K3-NBT reducÃ-ase in tissue sections.
Similar distribution and intensity were obtained using NADPH as
substrate instead of NADH.
In Chart 2, the quantitative histochemical results are pre
sented. For each area measured, an absorbance histogram
distribution was obtained. Then all measurements in all of the
sections studied were averaged for each class, giving the pop
ulation histogram presented in the graph. It can be observed
that the 3 different curves (for mucosa, superficial epithelium,
and tumors) all show similar absorbance for NADH and NADPH.
These curves also show that the tumors are nonhomogenous
with areas showing low and high absorbances. However, higher
absorption values (1.0 absorbance and more) are only observed
in the tumors.
The mean values of the absorbance measurements are pre
sented in Table 5. Again, it can be seen that the values for
NADPH and NADH are similar in the 3 different regions, except
in the superficial mucosa where the NADH values are higher than
the NADPH ones.
The correlation between the quantitative mean histochemical
absorbance values for the reduction of NBT by NADH and
NADPH is shown in Chart 3. Again, as with the biochemical
NADH deep mucoso
NADPH deep mucoso
NADH superficiol epithelium
NADPH superficiol epithelium
NADH tumor
NADPH tumor
.20
.40
.60
Quantitative cytochemistry
AbsorbanceLocationMucosa
±0.08a
(23)"
±0.01
(20)
0.34
±0.11
0.47 ±0.17
(21)
(19)
0.78 ±0.35
0.77 ±0.32
(26)NADPH-KS-NBT0.20 (25)
values plotted in Chart 1, there is a good correlation between
the reduction obtained with NADH and NADPH (r = 0.89), and
there are tumors with high and low activity. The number of
tumors with low histochemical activity is smaller than the number
of tumors with low biochemical activity. This difference is due to
the fact that the biochemical values represent an average; mean
while, the histochemical values were selected, and necrotic areas
were avoided.
BIOCHEMISTRY
o Mucoso
•Tumor
v
.140 .160 .180
a Mean ±S.D.
6 Numbers in parentheses, number of samples analyzed.
IOO
Q_
.120
Table 5
of the NADH- and NADPH-vitamin K3-NBT reducÃ-ase
TumorNADH-K2-NBT0.22
140
I 20
.IOO
DENSITY
Chart 2. Mean frequency distribution of absorbances of the NADH- and NADPHNBT reductases in different regions of the colon; abscissa, absorbance values;
ordinate, frequency distribution histogram. Lines join histograms of the frequency
distribution; Bars were omitted for clarity of the chart, and for each measurement
the computer offered a histogram distribution of the frequency of the points
measured; afterward, the different histograms were averaged for each case, and
all of the histograms were averaged again to obtain the composed histogram of
the population (lines).
Superficial Epithelium
I60r
.80
OPTICAL
80
CL
o
9 60
DISCUSSION
40
20
20
40
60
80
IOO
120
IO-I2M red.DCP¡P/NADPH/jugP/min
I40
I60
Chart 1. Correlation between the activities of the NADH-DCPIP and of the
NADPH-DCPIP reductases by the soluble fraction of normal colonie mucosa and
colonie carcinomas.
4852
Our results have shown that, in human colonie carcinomas,
the activity of the soluble enzyme DT-diaphorase is markedly
increased. In addition, we have shown that the histochemical
enzymes NADH- and NADPH-menadione-NBT reductases are
also increased in human colonie carcinomas and that the increase
in their histochemical activity is similar for both enzymes. There
fore, we believe that the histochemical enzymatic activity of our
histological sections may represent the real distribution of the
enzyme DT-diaphorase. Although the biochemical results are
CANCER RESEARCH VOL. 43
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Human Colon Cancer Enzymology
1.8 r
responsible for the histochemical distribution of the different
oxidative enzymes (6). The existence of a soluble NAD(P)Hdependent diaphorase was established later on (4).
Our results show that 70% of our cases show a high biochem
ical activity and that 75% show a high histochemical activity, but,
irrespective of the values (higher or lower activity), the values
with NADH always parallel the changes observed with NADPH.
The significance of the changes described in this paper will
not be clear until the function of the DT-diaphorase is completely
elucidated. The enzyme is induced in the liver and other organs
of experimental animals by polycyclic hydrocarbons (10, 13, 14,
19,23). The enzyme is a quinone reductase and, as such, seems
to intervene in the metabolism of polycyclic hydrocarbons by
reducing quinone derivatives of benzo(a)pyrene to hydroquinones prior to their conjugation to glucuronic acid derivatives
(13,14,16) and, recently, more evidence has been presented to
support the above hypothesis (15), and now it is postulated that
the DT-diaphorase may act as a free radical scavenger by
removing O2" generated by benzo(a)pyrene quiñonesformed by
1.6
a
1.4
z
HISTOCHEMISTRY
o Mucosa
P 1.2
i
|
•Tumor
1.0
o 0.8
û
< 0.6
«o,
•o
o o
t
0.2
0.2
0.4
0.6
0.8
1.0
1.2
1.4
Mean Absorban« NADPH-K3-NBT
1.6
Charts. Correlation between the mean peak histochemical absorbance of
NADH- and NADPH-NBT reductases by colonie mucosa and colonie carcinomas.
novel, the histochemical observations are not. Wattenberg (30)
reported that the histochemical activities of the enzymes NAD
and NADP diaphorase were elevated in colonie carcinomas and
observed a higher deposition of formazan when NAD was used.
He also observed that this increase was not homogeneous, that
some tumors showed a higher activity than others, and that
there were differences in intensity in the same tumors. He also
noted a higher activity of the enzyme in the superficial epithelium.
McGinty ef al. (17) also studied the tetrazolium reducÃ-asein 40
cases of colonie carcinomas, and found that the NADH diaphor
ase was elevated in 17 cases and that the NADPH diaphorase
was elevated in 15 cases. They also observed that the reaction
with NADH was greater than with NADPH. Later on, another
qualitative histochemical study (12) reported that 10% of the
cases showed an increase in the activity of the NADH diaphor
ase, and that 35% showed an increase in the activity of the
NADPH diaphorase. We believe that the lack of agreement
between our histochemical results and the ones already re
ported, namely, a higher activity of the NADH diaphorase over
that of the NADPH diaphorase and an unequal activation of both
diaphorases in the colonie carcinomas, is due to 2 factors: (a)
the use of formaldehyde vapors; and (b) the use of vitamin K3 in
the incubation media. Menadione has already been used for the
demonstration of the soluble tetrazolium reductase in the brain
(9), and formaldehyde vapors have been used for the demon
stration of soluble dehydrogenases in the intestines (8). A higher
rate of reduction of tetrazolium salts by NAD-dependent dehy
drogenases, when compared with the NADP-dependent ones,
has already been reported (6, 18). In all of these reports (6, 12,
17, 18, 30), neither menadione nor fixation by formalin vapors
was used. However, even the first report for the histochemical
activity of oxidative enzymes clearly showed that 2 diaphorases,
one dependent on NAD and the other dependent on NADP, were
OCTOBER
1983
the action of microsomal enzymes prior to the formation of
glucuronyl conjugates.
The increase in the activity of the enzyme can also be inter
preted in another way. The soluble DT-diaphorase may act as a
shuttle transferring electrons from NADH and NADPH to the
mitochondria! respiratory chain; the shuttles used for NADH
transfer are decreased in colonie carcinomas (2, 25). Evidence
for this function for the DT-diaphorase is meager (3). However,
it remains as a possibility, because coenzyme Q is decreased in
neoplasms (24, 27), and colonie carcinoma mitochondria are
functionally deficient (28). Therefore, it is possible that the in
crease in activity may represent an effort to compensate for the
loss of normal cellular respiratory functions.
Summarizing, our results have shown that the activity of the
enzyme DT-diaphorase is markedly elevated in colonie carcino
mas and that the increase in tetrazolium reduction in histochem
ical reactions observed by us and other workers (12, 17, 30)
may be the result of the activity of the enzyme. The biological
significance of these changes will remain undefined until the role
of the DT-diaphorase in the biology of the cancer cell can be
completely understood. The possibility of the function of the DTdiaphorase as a possible free radical scavenger is being investi
gated at this moment in our laboratory; if the results will support
this possibility, a case can be made that the function of the DTdiaphorase in colonie carcinomas is to help the survival of the
cancer cell when challenged with toxic agents. Similar function
has been postulated for the enzyme in rat liver tumors (21). It is
possible that the enzyme is part of the armory of emergent
resistant cells and of established neoplastia cells in their defense
against xenobiotic damage already observed in preneoplastic
and neoplastic lesions in rat liver (5).
ACKNOWLEDGMENTS
The authors are indebted to Dr. K. B. Farris and Dr. W. H. Luer (West Jefferson
Hospital); Dr. W. T. Mitchell, Dr. G. W. Fair, and Dr. G. W. Willis (Ochsner
Foundation Hospital); Dr. J. Skinner, Dr. J. Jarrel, and Dr. B. Faust (Southern
Baptist Hospital); and Dr. P. T. Riehl (Touro Infirmary) for help in obtaining the
surgical specimens. We also thank S. Giltespie for expert technical assistance.
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1
Fig. 1. Histochemical slaining of Ihe NADH-vilamin K3-NBT reducÃ-asein colonie
mucosal glandular epithelium, x 280.
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Human Colon Cancer Enzymology
Fig. 2. Histochemical staining of superficial epithelium by the NADH-vitamin K3NBT reducÃ-ase, x 280.
Fig. 3. Histochemical staining of colonie carcinomas by the NADH-vitamin «3NBT reducÃ-ase, x 280.
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Biochemical and Quantitative Histochemical Study of Reduced
Pyridine Nucleotide Dehydrogenation by Human Colonic
Carcinomas
Norberto A. Schor and Cornelis J. Cornelisse
Cancer Res 1983;43:4850-4855.
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