Thz JOURNAL OF HISTOCHEMISTRY AND Copyright © 1977 by The Histochemical

THE

CYTOCHEMISTRY Society,

Vol.

DISTRIBUTION OF ACID IN PRENEOPLASTIC

AND ALKALINE RIBONUCLEASE AND NEOPLASTIC RAT LIVERS’

S. M. MURTHY2 Inst

itut

du

Cancer

de Montr#{233}al, Centre

25, No. 2, pp. 115-121, 1977 Printed in U.SA.

Inc

ACTIVITIES

AND R. DAOUST3

Hospitalier

Not re-Dame,

and

D#{233}partement

d’Anatomie,

Universit#{233}

de

Montr#{233}al, Montr#{233}al, Canada Received

for publication

June

8, 1976,

and

in revised

form

September

28, 1976

Ribonuclease (RNase) activities revealed by the substrate film method were compared with reactions for acid and alkaline RNases obtained by lead precipitation technique in serial sections of preneoplastic livers and hepatomas. The preneoplastic parenchymal tissue giving positive reactions with ribonucleic acid films showed both acid and alkaline RNase activities by lead precipitation technique, and the areas of hyperplastic nodules nonreactive against substrate films were deficient in acid and alkaline RNase activities. Preneoplastic hyperbasophilic foci and hepatoma gave weak or negative reactions by either method, but necrotic areas and stromal tissue showed appreciable RNase activities. Thus a good correlation was observed in these tissues between the RNase activities revealed by the film method and those demonstrated by lead precipitation. Histochemical

investigations

by

strate film method revealed nuclease (RNase) activity ules of rats fed carcinogenic dimethylaminoazobenzene methylaminoazobenzene mine (7). Analyses

(2) of a large

neoplasms and tumors showed that neoplastic void of RNase activity the

necrotic

RNase vations

areas

represent

rats

receiving

further nant levels

a significant technique,

mucosa

in pre-

This

work Cancer

was

supported

Institute

by of Canada,

grants

from Ue

1560

est,

rue

Sherbrooke,

Montr#{233}al,

alkaline

RNase

the retain

malighigh

activities,

the

lead

analyses sections

a

the

technique

by the two of a particular

on agreefar to

different tissue.

to carry out such a livers to deter-

mine whether revealed by

of

the

the areas ribonucleic

correspond RNase actually

alkaline RNases elements giving strate films should activities by and negative

Minist#{232}re

precipitation

tissues are in good general no attempt has been made thus parallel in serial

alkaline films are

des Affaires sociales du Qu#{233}bec,La Fondation J.H. Biermans and Ues Fondations J. Rh#{233}aume. 2 Present address: Department of Immunochemistry Research, Evanston Hospital, Evanston, Illinois. Send reprint requests to: Dr. R. Daoust, Institut du Cancer de Montr#{233}al, Centre Hospitalier NotreDame,

and

to

necrotic authors

It was deemed of interest comparison with preneoplastic

always

during (8). They

a lack of both enzyme activities in cancer of various origins, whereas positive reac-

National

and

conduct methods

in

and alkalivers of and

of the gastric carcinogenesis

of acid

pathologic ment, but

Taper

a loss of acid in preneoplastic

for the stromal and 12, 14-16). These

reported that, contrary tumors, the benign tumors

method

obserRNase step

(8,

fact indicating that RNase deficiencies could be specifically associated with malignancy (14, 15). The results obtained by the substrate film

appreciable

N-nitrosomorpholine,

neoplastic lesions methylnitrosourea noted cells

exhibit

precipitation

et al. (16) demonstrated line RNase activities

tions were obtained tissues of tumors

(2), 3’-methyl-4-dior diethylnitrosavariety of human

(2, 3, 6, 7, 10, 11). These that a depletion in

activity might carcinogenesis. Using a lead

sub-

in experimental animals cells are generally dowhereas the stroma and

of tumors

activities suggested

the

a focal loss of riboin hyperplastic noddiets containing 4-

to areas activities, hydrolyzed of fresh positive exhibit

the lead reactions

RNase depletion acid film method

deficient

in acid

and

If ribonucleic by both acid

acid and

tissue sections (5), reactions with subacid and/or alkaline

precipitation in ribonucleic

technique, acid films

should coincide with areas showing low acid and alkaline activities. Other sets of results would necessitate a reevaluation of the methods

Canada. 115

such

com-

parative studies in order to better assess histochemical methods for RNase activities.

and

it seems

important

to pursue

the

116

MURTHY

AND

DAOUST

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RIBONUCLEASES MATERIAL

Animals:

AND

DURING

METHODS

without

Male

previously

described

(1).

Frozen

liver

and

alkaline

tion

technique.

RNase

activities

Acid and alkaline by the procedures

by

the

an

Taper

(13),

respectively.

livers

were

spread

Fresh on glass

frozen

slides,

following

media.

For

acid

RNase,

the

consisted of 25 ml of 0.2 M acetate buffer mg yeast ribonucleic acid (Schwarz/Mann), acid phosphatase ceuticals Inc.), ml

0.5

of distilled

from ml

water.

wheat of 0.4 For

germ

(ICN

alkaline

were

sulfide

carried

FIG.

1.

out

Liver

blue

showing

ing

parenchymal FIG.

stained

and with

Control incubated

tissue.

appreciable 3.

FIG.

4.

whereas

Alkaline intense

found

alkaline Livers zene: nodules

precipitation the

or

blue

incubated

for fed

and tant

surrounding with the

ules, liver trated

Pharmaand 74.5

in

the

con-

of

1 was

develop after

liver

2

1 represents with toluidine

nodule, and

a

trabeculae

connective

tissue,

tissue. Concomiof hyperplastic nod-

of RNase (2), and

deficient acid film

sub-

demonstration

Figure stained

parenchymal development

Figure

completely ribonucleic

without

hepatocarcinogen cells in the

ducts

a focal loss parenchyma

for alkaline obtained in

were observed in phosphatase. Mg2

a hyperplastic of bile

RNase

4-dimethylaminoazoben-

Rats fed this of parenchymal

showing

tissue

acid

in media

necessary

composed

technique.

positive reactions precipitation was

RNase. of rats

paren-

connective

negligible

to 3 months of feeding. portion of a liver section

medium

activity occurs in the nodule illus-

observed

to be

almost

in RNase activity when was exposed to an adjacent

a

serial section (Fig. 2). Surrounding parenchymal tissue showed appreciable activity. Comparable results were obtained with serial sections processed

for

alkaline tion

reactions in

intense

media

RNase

technique

plastic whereas

the

demonstration

activities (Figs.

3 and

nodule exhibited the surrounding reactions.

fixed of bile ducts

of

by the

lead

4). The

large

weak tissue gave

Consistent

in Carnoy and and connective

also

stained tissue,

acid

and

precipitahyper-

activities relatively

were

the

re-

with toluidine and surround-

x40.

acid film exposed positive reactions. activity

is observed

to adjacent Relatively in surrounding

Acid RNase activity demonstrated by the Sites of RNase activity are visualized by deposits hepatocytes of the hyperplastic nodule as compared FIG.

gave lead

sections

was

section from rat fed 4-dimethylaminoazobenzene a hyperplastic nodule, trabeculae composed

2. Ribonucleic areas indicate

whereas

mounted. sections

but No

liver

cytoplasm of hepatocytes for acid and alkaline and

weak

in

ex-

revealed

(2).

strate and only faint reactions the absence of exogenous

sisted of 50 ml of 0.05 M Tris-maleate buffer (pH 8.0), 20 mg yeast ribonucleic acid (Schwarz/Mann), 10 mg alkaline phosphatase from chicken intestine (Nutritional Biochemicals Corp.), 15 ml of 0.2% Pb(NO3), 35 ml of distilled water and 20 drops of 0.5 M MgCL After incubation the tissue sections were washed in distilled water, immersed in 1% acetic acid, washed again in water, developed in dilute ammonium

showed

liver

fixed

it

on

films

livers

activity

lead

canaliculi

activity RNase.

of rat

RNase,

the

rat

noted

and the reactions

by bile

cells

(pH 5.9), 20 10 mg

M Pb(N03)2

RNase

as previously

RNases

in either Baker’s formol-calcium for 15 mm or in acetone for 5 mm at 4#{176}C.The sections were then washed in distilled water and incubated for 3 to 6 hr in the

assays

acid

of normal

appreciable

The

localized (17) and and

control

Ribonucleic

to sections

The nuclei gave positive

toiui-

sections

thawed

livers:

chyma,

precipita-

RNase activities were described by Vorbrodt

phosphatases.

further

RNase.

posed

of acid

lead

exogenous

in

RESULTS

sections

with

or

omitted

Normal

blue.

Adjacent serial sections were stained dine blue or processed for the demonstration

was

alkaline

were deposited and thawed on ribonucleic acid films. After incubation at room temperature in a humidified atmosphere for 20 to 45 mm, the sections were removed by flushing with distilled water, and the ribonucleic acid films were stained directly with toluidine

substrate

MgCl2

albino rats of the Wistar strain (200-250 g) were fed a low protein diet (diet 3 of Miller et al. (9)) containing 0.06 % of 4-dimethylaminoazobenzene (Eastman Kodak Co.). Animals fed this diet and water ad libitum were killed by decapitation at various time intervals during a 180-day period of feeding. Sections of frozen liver tissue, 15 ,ti in thickness, were prepared in a cryostat at - 10#{176}C to -15#{176}C. Demonstration of RNase activities: The procedure for the demonstration of RNase activity by the substrate film method was essentially the same as that

117

HEPATOCARCINOGENESIS

RNase reactions

activity in adjacent serial are observed in surrounding

serial section and stained low RNase activity is shown parenchymal

lead precipitation of lead sulfide to that shown

tissue.

blue.

Unnodule

x 40.

technique precipitates. by surrounding

section. Weak activity nodules. x40.

with toiuidine by the hyperplastic

in adjacent serial section. Weak reaction given by tissue. x 40.

exhibited

by

the

large

nodule

.5... S #{149} #{149}

#{149}#{149}#{149}

.

#{149}

#{149}S

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5.

1. .5-i

b 0,

FIG. 5 and 6. Area of preneoplastic liver (Fig. 5) and well developed hepatoma (Fig. 6) showing intense hyperbasophiiia after toluidine blue staining in rats fed the carcinogenic azo dye. x40. FIG. 7 and 8. Acid RNase activities in adjacent serial sections. Weak activities are shown by hepatocytes of the hyperbasophiiic focus and the tumor but positive reactions correspond to necrotic areas. x 40. FIG. 9 and 10. Alkaline RNase activities in adjacent serial sections. Negative reactions given by the hyperbasophilic and tumor cells. High activity is noted however in stromai connective tissue of the hepatoma. x40.

118

,

-

.

.5

S#{149}’

I

11. Portion of a hepatoma including large necrotic zones. Section from azo dye-fed animal stained toluidine blue. x40. FIG. 12. Ribonucleic acid film exposed to adjacent serial section and stained with toluidine blue. Appreciable RNase activity is exhibited by a relatively large area showing necrotic alterations. x40. FIG. 13. Acid RNase activity in adjacent serial section. Higher activity is observed in the necrotic area than in the surrounding tumor cells. x40. FIG. 14. Alkaline RNase activity in adjacent serial section. Positive reactions given by necrotic cells and the stromai connective tissue. x40. FIG.

with

119

120

MURTHY

suits

for the

areas

of low

adjacent nodules. A more prolonged carcinogen renchymal ing

feeding

(Fig.

5). Such

regions

erties tumors

foci

also

are

acid considered

stration of acid RNase rial sections revealed

areas tissues.

positive

basic

to be the (4) and from

hyperbasophilic

hyperbasophilic against

focus

the of pastain-

with

prop-

foci ribonucleic

(2) except for necrotic and stromal often give positive reactions. The

hyperbasophilic and 8). The

in

with

transformation probably develop

exhibit

(Fig. 6). The are inactive

films which

activities

of rats

ribonucleic

actual sites of neoplastic the hepatomas that these

high

results in appearance of foci cells characterized by intense

of cytoplasmic

dyes

and

AND

and

the acid

tissues demon-

activity in adjacent low activity in both and reactions

the

tumor observed

were apparently associated Alkaline RNase activity

sethe

(Figs. 7 in some

with necrotic was almost

totally deficient in serial sections of the same tissues (Figs. 9 and 10). The stromal connective tissue of the tumors gave however strong reactions for alkaline RNase (Fig. 10). RNase crotic cated

activity

zones at the

showing

is commonly

of tumors periphery

signs

of

observed

in ne-

(3, 14-16). The cells loof the necrotic areas and

degeneration

are

especially

active, whereas the completely autolyzed tissue in central zone is usually negative (14-16). Figure 11 illustrates toluidine blue stained preparation from a portion of hepatoma with a large area film veals

of necrotic exposed to appreciable

area and

(Fig. 12 N), and alkaline RNase

creases necrotic

tissue (N). A ribonucleic acid an adjacent serial section reRNase activity in the necrotic parallel activities

in both enzyme zone of the tumor

analyses of acid indicate in-

activities mass

in (Figs.

the same 13 and 14

N).

DAOUST

be stressed zymes had sues,

the

however similar possibility

demonstrate RNase

activity on

before

drawing

areas

RNase neoplastic

clease activities, premalignant genesis

loose

(14)

of nucleases distinguish growth. The

Further necessary regard.

process, that the in liver

and the

the

fact tumors

nuclease

that experiretain nu-

problem

of

benign carcino-

activity,

leads

histochemical

represent benign the

in

of RNase

whereas histologically nodules in experimental

might between

the for-

plausible occurs

devoid

benign

that

the focal hyper-

the acid precedes

nodules

their

films

alkaline

in this

negative, it seems transformation

to suggest

entis-

foci and the developlatter tissues being

The

Furthermore, human

and

be

both change

of hyperplastic

activities. mental

out.

observed

involves This

of hyperbasophilic of hepatomas.

consistently that the

ruled would

conclusion

activity

nodules RNases.

mation ment

be

or

the carcinogenic better establish

of RNase

plastic alkaline

acid

acid

tissues firm

the two in these

ribonucleic

cannot

various

Concerning present results

per

that

preferentially

studies

loss

that because distributions

Ta-

analyses

a useful means and premalignant

spontaneous

to

“reactiva-

tion” of nucleases in necrotic portions of tumors is also of interest. The positive reactions for nucleases being associated mainly with early stages

of cell

necrosis

(14),

the

necrotic

process

occurring in portions of untreated tumors sents similarities with that induced by peutic agents. that therapeutic

Several agents

studies have raise the level

prethera-

indicated of tumor

RNase activities (whereas ineffective agents do not) and such increases precede obvious regression

(see

References

5). Activation

in

of RNases

thus seem to represent one of the early biochemical changes in tumor necrosis and it may possibly play a causal role in tumor regression.

DISCUSSION

A close correlation plastic and neoplastic activities and tion positive

revealed

those demonstrated technique. The reactions

showed both ties, and the

acid areas

was livers by the

observed between

substrate by

tissue with

in preneothe RNase film

the lead components

ribonucleic

and alkaline nonreactive

method precipitagiving

acid RNase against

ACKNOWLEDGMENTS

The Leblanc Mr. phy.

authors wish for valuable

Bernard

Szirth

cleic acid films gave little or no lead precipitation. The reactions obtained by substrate film method thus apparently represent the sum of acid and alkaline nuclease activities. It should

for

LITERATURE

films activiribonu-

to thank technical assistance

Miss Jeannine assistance and in photogra-

CITED

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1966

2. Daoust R: Focal loss of ribonuclease activity in preneoplastic rat liver. Cancer Res 32:2502, 1972 3. Daoust R, Amano H: Ribonuclease and deoxyri-

RIBONUCLEASES

4.

5. 6.

7.

8.

9.

10.

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piasiques 11.

DURING

Steigleder

dans

ies

tissus

normaux

et n#{233}o-

du tabac. Rev Can Biol 32:261, 1973 GK, Fisher I: Uber die Uokalization

121

HEPATOCARCINOGENESIS

von clease

Ribonuclease

(RNase)

und

-

(DNase)-Aktivit#{228}t

in

Deoxyribonu-

normaler,

in

ent-

zUndlich ver#{227}nderter Haut und bei Hauttumoren. Ark Kim Exp Dermatol 217:553, 1963 12. Taper HS: L’activit#{233} des nucl#{233}ases et phosphatases acides et alcalines dans les tumeurs r#{233}nales induites chez le rat par la dimethylnitrosamine (#{233}tudehistochimique). Pathoi Eur 2:406, 1967

13. Taper HS: La detection histochimique soxyribonuclease alcaline. Ann 14:301, 1968 14.

Taper

HS:

cal

activity

and

man.

The

relation

between

of nucleases These

and

d’agregation,

the

de la d#{233}Histochem histochemi-

neoplasms

in rat

Universit#{233}

catho-

lique de Uouvain, Vander, Louvain 1975 15. Taper HS, Brucher JM, Fort U: Activity of alkaline and acid nucleases in tumors of the human central

nervous

system

(histochemicai

study).

Cancer 28:482, 1971 16. Taper HS, Fort U, Brucher JM: Histochemical activity of alkaline and acid nucleases in the rat liver parenchyma during N-nitrosomorphoiine carcinogenesis. 17.

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Vorbrodt A: Histochemical studies on the cellular localization of acid deoxyribonuclease. Histochem Cytochem 9:647, 1961

intraJ