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[CANCER RESEARCH 50, 5112-5118, August 15, 1990]
Quantitative Cytochemical Detection of Malignant and Potentially
Malignant Cellsin the Colon1
Alistair James Best,2 Pranab K. Das,3 Hitendra R. H. Patel,2 and
Cornelis J. F. Van Noorden4
Laboratory of Cell Biology and Histology, University of
Amsterdam, Academic Medical Centre, Meibergdreef 15, 1105 AZ
Amsterdam, The Netherlands
ABSTRACT
It was found to be possible to distinguish malignant cells from
normalcells by using an oxygen-sensitive tetrazolium salt
(neotetrazolium) forthe histochemical demonstration of
glucose-6-phosphate dehydrogenaseactivity in cryostat sections of
human colon. We have studied 12 cases ofestablished adenocarcinoma
of the colon in addition to 4 of ulcerativecolitis and 4 of
adenomatous polyposis (polyposis coli). In a nitrogenatmosphere the
activities of malignant and normal cells were similar.However,
after incubation in an atmosphere of pure oxygen, only malignant
cells gave a positive reaction after 5 min. Three of the four cases
ofadenomatous polyposis gave a positive reaction for
glucose-6-phosphatedehydrogenase activity in oxygen in a manner
similar to that of specimenswith severe dysplasia. In general,
positive foci were histologically indistinguishable from the
neighboring tubuli. However, foci of severelydysplastic epithelium
usually showed a positive reaction. All three patients eventually
developed clear-cut severe dysplasia. The other patient,who showed
a negative reaction in oxygen, was diagnosed after 3 yearsas not
suffering from dysplasia. All cases of ulcerative colitis gave
areaction in oxygen comparable with that of normal cells.
Therefore, theareas with a positive reaction are considered to be
either in the processof malignant transformation or malignant. An
explanation for the oxygeninsensitivity of cancer cells appeared to
be a decrease in the activity ofSuperoxide dismutase (EC 1.15.1.1),
as addition of exogenous Superoxidedismutase to malignant cells
caused a normal reaction. We wish to suggestthat this test in
combination with the routine histology may be exploitedfor the
diagnosis of polyps in premalignant conditions.
INTRODUCTION
Because the process of carcinogenesis involves a
stepwiseprogression, which often takes over 20 years in humans (1),
theprospect of detecting at an early stage those cells that
willprogress to malignancy is important. Initiated cells often
formstructures considered to be precancerous, such as polyps
orpapillomas, some of which will eventually progress to malignancy
(2). Therefore a simple test that is able to distinguishsuch cells
at an early stage would be a valuable tool in diagnosis.However,
morphological changes often follow the events whichcause cells to
progress to malignancy (3); thus by the time a cellcan be
distinguished as malignant by morphological criteria itscancerous
nature is already well established.
Changes in the activities of certain enzymes occur at
earlystages of malignancy (4) and hence are detectable before
themorphological changes. One such enzyme is glucose-6-phos-phate
dehydrogenase (EC 1.1.1.49), the key regulatory enzymeof the
pentose shunt pathway that produces both NADPH andthe precursors of
nucleic acid synthesis (5) which are needed in
Received 10/18/89; revised 3/28/90.The costs of publication of
this article were defrayed in part by the payment
of page charges. This article must therefore be hereby marked
advertisement inaccordance with 18 U.S.C. Section 1734 solely to
indicate this fact.
1This study has been performed in the Laboratory of Cell Biology
andHistology, University of Amsterdam in conjunction with ERASMUS
programICP-89-UK-0172.
2 Department of Anatomy and Physiology, University of Dundee,
Dundee.
DD24HN, Scotland, United Kingdom.3 Department of Pathology,
Academic Medical Centre, Meibergdreef 15. 1105
AZ Amsterdam. The Netherlands.4 Laboratory of Cell Biology and
Histology, Academic Medical Centre, Mei
bergdreef 15, 1105 AZ Amsterdam, The Netherlands. To whom
requests forreprints should be addressed.
large amounts by malignant cells.Demonstration of this enzyme is
possible in tissue sections
by using a tetrazolium salt coupled to an intermediate
electroncarrier. Production of an insoluble colored formazan marks
theareas of enzyme activity (6). Oxygen present in the
immediatereaction area is able to interfere with the production of
formazan if either low concentrations of the tetrazolium salts
(
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CYTOCHEMICAL DETECTION OF MALIGNANCY IN THE COLON
temperature was —¿�25°C;the sections were placed on clean
glass slides
and stored in the cryostat cabinet until use (1 to 2 h). Serial
sections tothose used for enzyme histochemical studies were also
stained withhematoxylin and eosin for reference to all
formalin-fixed tissue.
The incubation media for glucose-6-phosphate dehydrogenase
consisted of: 50 mM glycyl glycine buffer (BDH Chemicals, Poole,
Dorset,United Kingdom) (pH 8.0) or 100 mM phosphate buffer (pH
7.45)containing 20% (20 g/100 ml) polyvinyl alcohol (hot water
soluble;average molecular weight 40,000; Sigma Chemical Co., St.
Louis, MO)(15), 10 mM glucose 6-phosphate (Boehringer, Mannheim,
FederalRepublic of Germany), 0.8 mM NADP (Boerhinger), 4.5 mM
neotetra-zolium chloride (Polysciences, Northampton, United
Kingdom, orSigma; purified with chloroform) and 0.67 mM thionine
(Merck,Darmstadt, Federal Republic of Germany) as an alternative
electroncarrier to 1-methoxyPMS (16). The incubation medium for the
demonstration of NADPH dehydrogenase was identical but 0.67 mM
men-adione (sodium salt; Sigma) replaced thionine as the
intermediateelectron carrier (16, 17). In the course of the
investigation the followingcompounds were added to the basic
incubation media either separatelyor in combination: 1 mM dicumarol
(Sigma); SOD (Sigma; from bovineliver, 4300 lU/mg; 0.3 mg/ml
incubation medium); catalase (Sigma;from bovine liver, 1600 lU/mg;
0.1 mg/ml incubation medium); 10mM sodium azide (Merck).
The reaction was performed in a room maintained at 37°Con
the
stage of a Vickers M85a scanning and integrating cytophotometer
toobtain kinetic measurements (14). Polyvinyl alcohol-containing
buffer(5 ml) was placed in a tonometer (also at 37°C),an
instrument used to
equilibrate a liquid with a gas without forming bubbles (18),
and wassaturated with the appropriate gas (either nitrogen or
oxygen) at a flowrate of 500-800 ml/min for a minimum of 10 min.
Other constituentsof the basic incubation medium were then added.
This ensured thoroughmixing of the incubation medium in addition to
complete saturationwith either nitrogen or oxygen.
The area to be measured was selected and a drop of
incubationmedium on a cover slip placed over the section, any
readjustmentsneeded were then made. The first measurement was taken
at 30 s (timezero; the value measured also was readjusted to zero).
Measurementswere then taken every 30 s thereafter for periods of up
to 15 min. Allmeasurements were made at 585 nm, the isosbestic
wavelength for theformazans of neotetrazolium (19). A Leitz NPL x25
objective lens(numeric aperture 0.50) was used for the
measurements. The mask sizeused was Al (10 ¿imdiameter), giving an
effective scanning area of78.5 Mm2.A spot diameter of 0.8 Mmwas
selected, with a band width
setting of 65. Usually at least three kinetic measurements were
takenfrom each tissue sample in serial sections, from areas of both
high andlow activity. The same areas were then measured in further
serialsections using an incubation medium saturated with the
alternative gas.All integrated absorbance values were converted to
mean integratedabsorbance (19) and then to nmol of NADPH formed/mm3
of tissue
by the method of Van Noorden and Butcher (20). The mean
valuescalculated from the three kinetic reactions performed on each
tissuesample were then plotted and the rate of reaction was
calculated usingthe linear part of the graph, usually at 5 min. The
calculation of residualactivities was achieved by comparing the
actual mean activities obtainedafter a time of 5 min (ratio of the
activity in oxygen and in nitrogen)rather than the rates; this was
found to give more accurate results.
RESULTS
The histochemical method for the detection of
glucose-6-phosphate dehydrogenase activity was specific, inasmuch
as thecontrol reaction performed by the omission of the coenzymeand
substrate from the incubation media (21) reduced theproduction of
formazan to unmeasurable levels.
The use of phosphate buffer resulted in a lag phase of 4 minon
the average in normal tissue when the incubation was performed in
oxygen. On the other hand, glycyl glycine buffer gavea lower rate
of reaction and a longer lag phase in oxygen innormal tissue
(approximately 8 min). Therefore it was a distinct
advantage in showing the differences between malignant andnormal
cells to use glycyl glycine buffer. In human colonietissue formazan
formation was selectively prevented in cancercells in oxygen for
approximately 1.5 ±1.1 min (SD) (Table 1)and in normal cells in
oxygen for approximately 7.5 ±3.4 min(Table 2; Fig. 1). Of even
greater importance was the purity ofthe neotetrazolium used.
Neotetrazolium from Sigma was heavily contaminated with impurities
(22) that caused formazanformation in normal tissue in oxygen,
hence making it impossible to locate areas of malignant cells.
Sigma neotetrazoliumtherefore had to be purified with chloroform
prior to use (22).The batches obtained from Polysciences were found
to be pureenough to be used without chloroform treatment and were
usedfor all studies thereafter.
Carcinoma Cells. Carcinoma cells in human colon displayedvarying
activities of glucose-6-phosphate dehydrogenase (Table
1). The lag phase before any formazan formation was observedin
carcinoma cells was typically below 1 min when the reactionwas
performed in nitrogen (mean, 0.4 min). In oxygen the lagwas
slightly increased to 1 or 2 min (mean, 1.5 min).
The rate of formazan formation was not seriously affected bythe
presence of oxygen in the majority of cases (Table 1; Fig.2a). The
residual activity observed in malignant cells was between 30 and
over 100% ofthat in nitrogen (mean, 59%; Table1; Fig. 1) after 5
min incubation. No significant difference wasobserved between the
reaction rates in malignant cells in nitrogen compared to oxygen.
In nitrogen, a linear response wasobserved up to 7 to 11 min, in
oxygen the linear response wasusually for a longer period of time
(up to 15 min of incubation).No relationship was observed between
the degree of differentiation of malignant cells and the lag phase
or rate of reaction.
Normal Cells. In nitrogen, the lag phase exhibited by
normalcells was below 3 min (mean, 1.0 min), in oxygen this
wasextended over a range with a mean value of 7.8 min (Table 2;Fig.
2b). The difference between the lag phase for normal cellsin
nitrogen and in oxygen was significant (P < 0.001). Anyreaction
observed in normal cells before 5 min had elapsed wasalways lower
than 10% when compared to the reaction innitrogen (mean, 1.2%; P
< 0.001 compared to malignant cells;c.f. Tables 1 and 2). Hence
normal cells could still easily bedistinguished from malignant
cells by comparison of the reaction after 5 min in oxygen with that
obtained in nitrogen (Fig.1). The reaction rate at 5 min was always
considerably lower inoxygen than nitrogen (P < 0.001; Table 2;
Fig. 2b).
Adenomatous Polyps. All cases of adenomatous polyposiswere
classified independently in serial sections stained withhematoxylin
and eosin and were of varying grades. Resultsobtained with the
glucose-6-phosphate dehydrogenase reactionare shown in Table 3 and
Fig. 3. Those areas classified asnormal colonie tissue all showed
no formazan formation inoxygen (negative reaction). Some areas of
cells showing anegative reaction were classified by the pathologist
as dysplas-tic, whereas other areas showed a high activity in
oxygen(positive reaction; Table 3). These latter areas were
classifiedby the pathologist as severely dysplastic but were
morphologically difficult to discriminate from the areas of
dysplasia showing a negative reaction on frozen sections (Fig. 3).
On the otherhand, grading of dysplasia is often subjective when
using hematoxylin and eosin-stained sections of formalin-fixed
tissue.
Positive reactions were typically seen either in areas
composedof many glands showing a high reaction and separated
fromsurrounding structures by connective tissue, or in
singularglands surrounded by areas displaying very low reaction or
no
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CYTOCHEMICAL DETECTION OF MALIGNANCY IN THE COLON
Table l Glucose-6-phosphale dehydrogenase activity in malignant
epithelial cells in cases of adenocarcinoma of the colonThe
incubation was performed with neotetrazolium in nitrogen (N2) and
oxygen (O2) at 37°Cfor 15 min. Residual activity is the ratio of
mean activity from three
kinetic reactions in N2 and in ( )- after 5 min.
Rate at 5 min (fromlinear reaction(MMAgePatient
(yr)1
852753534605716627638759
831071118412
62Lag
period(min)SexFemaleMaleFemaleFemaleMaleMaleFemaleMaleFemaleFemaleMaleMaleN200000000.511110,0.50.50.50.50.51.520.522.533.5mm3N29.033.011.114.027.112.08.011.09.35.56.35.8min')0211.330.212.37.218.67.06.210.05.44.86.05.0Residualactivity
(%)1127872675133438757493029
Mean valuesLag phase
In N2: 0.4 ±0.5 minIn O2: 1.5 ±1.1 min
Reaction rateIn N2: 12.7 ±8.6 nM mm"3 min~'In O2: 10.3 ±7.4 nM
mm"3 min"'
Residual activity 59.0 ±25.3%
Table 2 Glucose-6-phosphate dehydrogenase activity of normal
colonie epithelial cells
Experimental conditions as for Table
Rate at 5 min (fromlinear reaction(nMAgePatient
(yr)1
852753534605716627638759
831071118412
62Lag
period(min)SexFemaleMaleFemaleFemaleMaleMaleFemaleMaleFemaleFemaleMaleMaleN2110.51.52000.5111.52.5024.594111056.53.57.58159mm'
min')N28.311.78.93.74.58.07.75.56.86.76.35.3020.700.600011.50000Residualactivity
(%)4.80100018.30000
Mean valuesLag phase
In N¡:1.0±0.8 minIn O2: 7.8 ±3.4 min
Reaction rateIn N2: 6.9 ±2.2 nM mm"3 min"In O3: 0.2 ±0.5 nM
mm"3 min"
Residual activity: 1.2 ±2.6%
reaction at all in oxygen (Fig. 3¿>).In nitrogen these
activitieswere very similar.
Cases of Ulcerative Colitis. Table 3 also shows the meanresidual
activity in epithelial cells affected by ulcerative colitis.None of
the cells exhibited any morphological signs of malignancy and
reacted in a manner which would classify them asnonmalignant
according to the present oxygen sensitivity test.No reaction was
seen in the connective tissue or, more importantly, in the cellular
infiltrate if thionine was used in theincubation medium. On the
other hand, when 1-methoxyPMSwas used instead of thionine, a very
high reaction was found ininflammatory cells in oxygen and
therefore could not be usedto indicate areas of malignancy.
Elucidation of the Mechanism of the Oxygen
InsensitivityPhenomenon. By replacing 1-methoxyPMS or thionine in
theincubation media with menadione, the activity of the enzymeNADPH
dehydrogenase is revealed (Fig. 4a). The reaction wasspecific
inasmuch as addition of dicumarol, a specific inhibitor
of this enzyme (23), reduced formazan production to low
levels.The effect of oxygen upon the residual activity of this
enzymein malignant cells was far greater than the corresponding
reaction for glucose-6-phosphate dehydrogenase activity (14%
com
pared with 31%). Addition of dicumarol to the incubationmedium
for glucose-6-phosphate dehydrogenase resulted in a
small but significant increase in the reaction (Fig. 4a).The
effect of exogenous SOD and catalase upon the residual
activity of malignant cells in oxygen is shown in Fig. 4b.
Adecrease from 31% activity to 5% activity was noted; i.e.,
themalignant cells showed the characteristic residual activity
displayed by normal cells in oxygen when exogenous SOD wasadded to
the medium. Addition of azide, an inhibitor of catalase(12),
increased the reaction in normal cells in oxygen (notshown). The
reaction was dependent upon an intermediatecarrier in the medium as
it was reduced to low levels when 1-
methoxyPMS or thionine was omitted (Fig. 5).5114
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CYTOCHEMICAL DETECTION OF MALIGNANCY IN THE COLON
Fig. 1. Serial cryostat sections (8 /an thick) of adenocarcinoma
of the colon,a, hematoxylin and eosin staining of normal (A/) and
malignant (CA) tissue; b,glucose-6-phosphate dehydrogenase activity
after 5 min of incubation usingneotetrazolium in an atmosphere of
nitrogen. Formazan is deposited in bothnormal (N) and malignant
(CA) tissue; c, as for b, but reaction was performed inan
atmosphere of oxygen, showing formazan deposition only in malignant
cells(CA).Bar, lOO^m.
150-
IOD
50-
IO 15
time
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CYTOCHEMICAL DETECTION OF MALIGNANCY IN THE COLON
4*'^
^ :•>.C;••¿�*
Fig. 3. Serial cryostat sections (8 ¿2Lc20-ËaT3
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CYTOCHEMICAL DETECTION OF MALIGNANCY IN THE COLON
15-
E
I10-
«PMS
•¿�oIE5Õ
5-o.OzsCn»PMS-PMS-PMS1
1
t umour/Anormal '
Fig. 5. Glucose-6-phosphate dehydrogenase activity (4- PMS) and
the proportion of NADPH utilized in cellular detoxification
reactions (—PMS; type Ireaction) in samples of adenocarcinoma of
the colon (tumor) and normal colonieepithelial cells (normal). The
data are expressed as nin NADPH formed/mm3 of
tissue/min. Type I activity in malignant tissue is not increased
in proportion tototal glucose 6-phosphate activity.
oxygen upon the formation of formazan from triphenyl
tetra-zolium has already been documented (26, 27). For
nitrobluetetrazolium (NBT), which has a lower electron potential,
areversible reaction occurs with atmospheric oxygen, again withthe
production of oxygen radicals (Reaction B) (7, 11,
28).Neotetrazolium does not show the same reversible
reaction,increasing the concentration of neotetrazolium in the
incubation medium does not decrease the effect of oxygen (28,
29).Only when all the oxygen has been removed will the
reactionproceed ( 18).
With respect to the initial formation of formazan exhibitedby
carcinoma cells in an atmosphere of oxygen (the oxygeninsensitivity
phenomenon), a factor is responsible for the increased reaction in
these malignant cells. A comparison ofTables 1 and 2 shows that
carcinoma cells may display a rateof reaction similar to that of
normal cells in an atmosphere ofnitrogen but exhibit a
significantly higher rate of reaction thannormal cells in oxygen (P
< 0.001). Without the addition of 1-methoxyPMS to the incubation
medium the proportion ofNADPH utilized for detoxification reactions
is revealed (6, 29).These reactions involve the enzymes NADPH
cytochrome P-450 reducÃ-ase and NADPH dehydrogenase. Since
NADPHcytochrome P-450 reducÃ-asehas been indicated as the
factorresponsible for the oxygen insensitivity phenomenon (30)
theactivity of this pathway was investigated. Fig. 5 shows that
thisenzyme accounts for only a small proportion of the
overallreaction in both normal and malignant cells (reaction in
theabsence of PMS). Moreover it is not known as a
transformation-linked enzyme (31) and has also been shown to be
oxygensensitive (32). We therefore conclude that this enzyme is
notinvolved in the oxygen insensitivity phenomenon in
malignantcells. On the other hand NADPH dehydrogenase is
transformation linked (17) but Fig. 4 shows that the reaction for
thisenzyme is oxygen insensitive only to a limited extent in
malignant cells but not to the extent that the reaction for
glucose-6-phosphate dehydrogenase activity appears to be.
Furthermorethe reaction for glucose-6-phosphate dehydrogenase is
not di-cumarol sensitive. The slightly increased reaction is
probably
due to the inhibition of endogenous electron transport pathways.
This evidence suggests that oxygen insensitivity is notmediated by
NADPH dehydrogenase but occurs at the level ofthe neotetrazolium
radical. This is also indicated by the factthat oxygen sensitivity
in normal cells has been observed whilestudying succinate
dehydrogenase activity in normal rat liver(18), suggesting that
oxygen insensitivity in malignant cells isindependent of NADPH.
Within certain limitations the enzymeused to demonstrate oxygen
insensitivity is in itself unimportant.
SOD is involved directly in the inhibition of formazan
production, inasmuch as its addition to the incubation mediumcaused
a decreased formation of formazan in malignant cells inoxygen (Fig.
4b). It is known that the activity of the manganese-SOD is absent
in carcinoma cells, whereas the activity ofcopper/zinc-SOD is
sometimes lowered (12, 33). The effect ofSOD on
neotetrazolium-formazan formation cannot be explained in the same
way as for nitroblue tetrazolium, where theequilibrium reaction
(Reaction B) is affected. Increasing theconcentration of
neotetrazolium did not affect the oxygen insensitivity of normal
cells and therefore Reaction A is not anequilibrium. Thus it is not
the removal of oxygen radicals thatis important here but the
reformation of molecular oxygen inthe reaction area. This reformed
oxygen can then react repeatedly with neotetrazolium radicals. In
the normal cell, the dis-mutation of oxygen radicals is extremely
rapid due to thepresence of SOD. This accelerates the normal
dismutationreaction by a factor of xlO4 at physiological pH (7.45)
(12). In
malignant cells the dismutation reaction is much slower due
todecreased SOD activity and is in competition with other reactions
undergone by the Superoxide radical that do not producemolecular
oxygen (12, 34). Thus in a malignant cell oxygen isremoved from the
reaction area within approximately 1 min,depending on the residual
levels of SOD present. Formazanformation then occurs at a rate
similar to that seen in nitrogen(Table 1).
Catalase (CAT) is able to enhance the reaction of SOD intwo
ways: (a) under the influence of catatase three-fourths ofthe
Superoxide radicals are converted into molecular oxygeninstead of
only one-half by SOD alone (Reactions C and D);(b) catatase removes
hydrogen peroxide formed by SOD; it isknown that hydrogen peroxide
inhibits copper/zinc-SOD (35).
2Or + 2H+ H2O2 + 02
CAT2H2O2 »2H2O + O2
(C)
(D)
Addition of azide to the incubation media reduces the lag
phasein normal cells by inhibiting catalase, thereby affecting
theefficient functioning of SOD. Catalase and SOD have beenshown to
be mutually supportive to each other (36). Once theinitial oxygen
has been removed the reaction proceeds unhindered with the
formation of formazan as shown in Reaction E.
NT—FT + NT—H' -> NT + NT—H2(formazan) (E)
If oxygen is recycled in the manner described here, a
relativelysmall proportion of oxygen can exert an effect much
greaterthan the absolute amount alone could account for. This
hasbeen documented with regard to fatty acid oxidation, the
exaggerated effect of oxygen being explained in a manner similarto
that proposed here (37).
In conclusion it is stated that the oxygen insensitivity
ofmalignant cells is caused by a decrease in cellular activity
of
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CYTOCHEMICAL DETECTION OF MALIGNANCY IN THE COLON
SOD. This prevents the recycling of molecular oxygen,
thusallowing the deposition of formazan within the first 5 min
ofincubation. Because levels of SOD are decreased in the
earlystages of malignancy and remain depressed (13), the
detectionof cancerous cells before morphological parameters
becomeapparent is a possibility as was shown with the four cases
ofcolonie polyps studied here. Therefore, the oxygen
insensitivitytest could be a useful additional tool for grading of
dysplasia.
The present study is based on cytophotometric analysis ofthe
reactions to firmly establish the validity of the test. However,
cytophotometry is not an essential tool for the test andmicroscopic
inspection of serial sections after 5 min of incubation in nitrogen
and oxygen is sufficient to detect malignantor potentially
malignant cells. False positive reactions have notbeen found thus
far in the present study when thionine wasused as exogenous
electron carrier. Inflammatory cells showedsome positive reaction
in the presence of oxygen when thioninewas replaced by
1-methoxyPMS. Other studies on the basis ofthe oxygen insensitivity
phenomenon (3, 8, 9, 10, 30) did notreport false positive findings
either.
ACKNOWLEDGMENTS
The authors would like to express their gratitude to Dr.
NormanWalfoord for his help in diagnosis of specimens. They would
also liketo thank the Pathology Department, Academical Medical
Centre, forsupply of the specimens. Thanks also to J. Peeterse for
photographicwork and to Professors J. James and P. J. Stoward for
making thiswork possible.
REFERENCES
1. Farber, E. Pre-cancerous steps in carcinogenesis their
physiological adaptivenature. Biochim. Biophys. Acta, 738: 171-180,
1984.
2. Farber, E. Chemical carcinogenesis: a current biological
perspective. Carcinogenesis (Lond.), 5: 1-5, 1984.
3. Heyden, G. Histochemical investigation of malignant cells.
Histochemistry,59:327-334, 1974.
4. Bannasch, P., Moore, M. A.. Klimek, F., and Zerban, H.
Biological markersof preneoplastic foci and neoplastic nodules in
rodent liver. Toxicol. Pathol-,10: 19-34, 1982.
5. Evans, A. W.. Johnson, N. W., and Butcher. R. G. A
quantitative cytochem-ical study of three oxidative enzymes during
experimental oral carcinogenesisin the hamster. Br. J. Oral Surg.,
IS: 3-16, 1980.
6. Van Noorden, C. J. F. Histochemistry and cytochemistry' of
glucose-6-phosphate dehydrogenase. Prog. Histochem. Cytochem.,
/5:1-84, 1984.
7. Van Noorden, C. J. F., and Butcher, R. G. The involvement of
Superoxideanions in the nitro blue tetrazolium chloride reduction
mediated by NADHand phenazine methosulphate. Anal. Biochem., 176:
170-174, 1989.
8. Ibrahim. K. S., Husain, O., Bitensky, L., and Chayen, J. A
modified tetrazolium reaction for identifying malignant cells from
gastric and coloniecancer. J. Clin. Pathol.. 36: 133-136, 1983.
9. Petersen, O. W., Hoyer, P. E., Hilgers. J.. Briand. P., and
Van Deurs. B.Characterization of epithelial cell islets in primary'
monolayer cultures ofhuman breast carcinomas by the tetrazolium
reaction for glucose-6-phosphatedehydrogenase. Virchows Arch. B
Zellpathol.. SO:27-42, 1985.
10. Butcher, R. G. The oxygen insensitivity phenomenon as a
diagnostic aid incarcinoma of the bronchus. In: J. R. Pattison. L.
Bitensky, and J. Chayen(eds.), Quantitative Cytochemistry and Its
Applications, pp. 241-251. NewYork: Academic Press, 1979.
11. Ponti, V., Dianzani. M. U., Cheeseman, K., and Slater, T. F.
Studies on the
reduction of nitro blue tetrazolium chloride mediated through
the action ofNADH and phenazine methosulphate. Chem.-Biol.
Interact., 23: 281-291,1978.
12. Halliwell, B., and Gutteridge, J.M.C. Free Radicals in
Biology and Medicine.Oxford, United Kingdom: Oxford University
Press, 1985.
13. Oberely, L. W., and Buettner, G. R. Role of Superoxide
dismutase in cancer:a review. Cancer Res., 39: 1141-1149, 1979.
14. Van Noorden, C. J. F. Enzyme reaction rate studies in tissue
sections. Proc.R. Microsc. Soc., 23: 93-97, 1988.
15. Van Noorden, C. J. F., and Vogels, I. M. C. Polyvinyl
alcohol and othertissue protectants in enzyme histochemistry: a
consumer's guide. Histochem.J., 21: 373-380, 1989.
16. Butcher, R. G., and Evans, A. W. Diffusion during
dehydrogenase reactions:the effects of intermediate electron
acceptors. Histochem. J., 16: 885-895,1984.
17. Schor, N. A., and Cornelisse, C. J. Biochemical and
quantitative histochem-ical study of reduced pyridine nucleotide
dehydrogenation by human coloniecarcinomas. Cancer Res., 43:
4850-4855, 1983.
18. Butcher, R. G. Oxygen and the production of formazan from
neotetrazoliumchloride. Histochemistry. 56: 329-340, 1978.
19. Butcher, R. G. Precise cytochemical measurement of
neotetrazolium formazan by scanning and integrating
microdensitometry. Histochemistry, 32:171-190, 1972.
20. Van Noorden, C. J. F., and Butcher, R. G. The out-of-range
error inmicrodensitometry. Histochem. J., IS: 397-398, 1986.
21. Butcher, R. G., and Van Noorden, C. J. F. Reaction rate
studies of glucose-6-phosphate dehydrogenase in sections of rat
liver using four differenttetrazolium salts. Histochem. J., 17:
993-1008, 1985.
22. Altman, F. P. Tetrazolium salts—a consumer's guide.
Histochem. J., 8: 471-
485, 1976.23. Dixon, M., and Webb, E. Enzymes, Ed. 2, London:
Longmans, 1964.24. Bodmer, W. F., Bailey, C. J., Bodmer, J.,
Bussey, H. J. R., Ellis, A., Gorman,
P., et ai. Localization of the gene for familial adenomatous
polyposis onchromosome 5. Nature (Lond.), 328:614-616, 1987.
25. Solomon, E., Voss, R., Hall, V., Boomer, W. F., Jass, J. R.,
Jeffreys, A. J.,et al. Chromosome 5 alÃ-eleloss in human colorectal
carcinomas. Nature(Lond.), 328: 616-619, 1987.
26. Sato. S. Free radicals in NADPH-microsomes—Triphenyl
tetrazolium chloride system as evidence by initiation of sulphite
oxidation. Biochim. Biophys.Acta, 143: 554-561, 1967.
27. Sato, S., and Iwaizumi, M. Free radical mechanism by which
triphenyltetrazolium chloride stimulates aerobic oxidation of NADPH
by microsomes.Biochim. Biophys. Acta, 172: 30-36, 1969.
28. Van Noorden, C. J. F. On the role of oxygen in dehydrogenase
reactionsusing tetrazolium salts. Histochem. J., 20: 587-593.
1988.
29. Altman, F. P. Quantitative dehydrogenase histochemistry with
special reference to the pentose shunt dehydrogenases. Prog.
Histochem. Cytochem., 4:225-273, 1972.
30. Petersen, O. W., Hoyer, P. E., and Van Deurs, B. Effect of
oxygen on thetetrazolium reaction for glucose-6-phosphate
dehydrogenase in cryosectionsof human breast carcinoma, fibrocystic
disease and normal breast tissue.Virchows Arch. B Zellpathol., 50:
13-25, 1985.
31. Buchmann, A., Kuhlmann. W.. Schwarz. M.. Kunz, W., Wolf, C.
R., Moll,E., et al. Regulation and expression of four cytochrome
P-450 isoenzymes,NADPH cytochrome P-450 reducÃ-ase,the glutathione
transferases B and Cand microsomal epoxide hydrolase in
preneoplastic and neoplastic lesions inrat liver. Carcinogenesis
(Lond.), 6: 513-521, 1985.
32. Altman, F. P., Bitensky, L., Butcher, R. G.. and Chayen. J.
Integrated cellularchemistry applied to malignant cells. In: D. M.
D. Evans (ed.), CytologyAutomation, pp. 82-97. Edinburgh: E. &
S. Livingstone, 1970.
33. Dionisi, O., Galeotti, T., Terranova, T., and Azzi, A.
Superoxide radicalsand hydrogen peroxide formation in mitochondria
from normal and neoplastic tissues. Biochim. Biophys. Acta, 403:
292-300, 1975.
34. Freeman, B. A., and Crapo, J. D. Biology of a disease. Free
radicals andtissue injury. Lab. Invest., 47: 412-426, 1982.
35. Hodgeson, E. K., and Fridovich, I. The interaction of bovine
erythrocyteSuperoxide dismutase with hydrogen peroxide:
inactivation of the enzyme.Biochemistry. 14: 5294-5299, 1975.
36. Geerts, A., and Roels, F. In vivo co-operation between
hepatic catalase andSuperoxide dismutase demonstrated by
diethyldithiocarbamate. FEBS Lett.,140:245-247, 1982.
37. Fried, R., Fried, L. W., and Babin, D. R. Biological role of
xanthine oxidaseand tetrazolium-reductase inhibitor. Eur. J.
Biochem., 33: 439-445, 1973.
5118
on June 26, 2021. © 1990 American Association for Cancer
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1990;50:5112-5118. Cancer Res Alistair James Best, Pranab K.
Das, Hitendra R. H. Patel, et al. Potentially Malignant Cells in
the ColonQuantitative Cytochemical Detection of Malignant and
Updated version
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