Ferritin is increased in diethylnitrosamine - altered rat hepatocytes

BIOCHEMICAL

Vol. 99, No. 3,1981 April

AND

BIOPHYSICAL

RESEARCH

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15, 1981

FERRITIN

IS

Carole

INCREASED

IN

Beaumont,

Elisabeth

Marie-France

Unit6

DIETHYLNITROSAMINE

Latinier,

de Recherches

January

Bourel

Hkpatologiques,

RAT

Christiane

Rumeur*,

Michel

35011

Received

Le

- ALTERED

INSERM

and

Guillouzo, Guillouzo

And&

49,

U

HEPATOCYTES

H6pital

de Pontchaillou,

- France

RENNES

14,1981

Summary : Ferritin was localized by immunoperoxidase in rat liver during the early stages of experimental carcinogenesis induced by diethylnitrosamine. Carcinogen - altered hepatocytes, identified by their reduction in membranebound ATPase activity, showed a highly elevated content in ferritin (or ferritin subunits), compared to normal hepatocytes. This could correspond to an accumulation of free subunits in the cytoplasm or to a “non-specific” increase in ferritin synthesis related to the carcinogenic process. Our results suggest that some cytoplasmic proteins other than enzymes can be modified during the early stages of carcinogenesis and that ferritin accumulation detedted by immunolocalization can be used as a valuable marker to identify foci of cellular alterations. Introduction variety

A

of cellular

before

cinogens

hepatocellular

tic

nodules

are

and

several

enzyme

for

the

their

two

has

lesions

are resistant

in

storaye could

the

*

during Supported

Abbreviations

this

by the

(6,

the

“Ligue

normal 9)

which

observations stages

we performed stages

of

neoplas-

heterogeneity all

has

been

shown

FranGaise

diethylnitrosamine ; SDS, PAP, peroxidase - antiperoxidase HEPES, N-2 Hydroxyethylpiperazine

it

sodium

an

in-

iron-

ferritin

synthesis In

order in rat

by diethylnitrosamine. le cancer”. dodecyl

sulfate

-N’-2-ethanesulfonic 0006-291

879

in

cellular

of ferritin

induced

the

with

has been well results

the major

contre

that

hand,

that

in

hepatocellular

is overloaded

immunolocalization

: DENA,

2)

markers

liver

suggest

(1,

valuable

4).

hepatocytes is

and

of hepatocarcinogenesis.

carcinogenesis

Nationale

it the

car-

chemical

described as

identifies

On the other

(8,

early

used

(3,

which

when

7).

foci

phenotypic

procedure,

into

ferritin

different

hypothesis,

the first

this

incorporation

during

cells

accumulation

by

precisely

been

have been

a new marker

With

of

These

modified

to verify liver

synthesis

protein. be

(51,

to iron iron

have

liver

Altered

considerable

display

administration that

that

reactions

developped.

dietary

demonstrated crease

may

properties been

lesions

in rat

develop.

carcinogen-altered

of

lesions by

carcinomas of

cells

these

enzymatic

iron

types

are produced

histochemical

identification

Since

lesions

;

acid

X/81/070879-07$01.00/0

Cop.vrigh/ G I981 by Academic Press, Inc. Ail righ/s of reproduction in any form reserved.

Vol.

99, No.

Material

3,198l

and

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

methods

Material

tial tube,

after trols.

Young adult female Sprague-Dawley hepatectomy, the rats received 24h, 48h and 72h later. Fifteen DENA treatment and 5 female

Immunological

rats were used. After two-third parthree doses of DENA (25 mg/kg) by stomach rats were sacrified at 3 and 6 months rats of the same strain were used as con-

methods.

Ferritin was prepared from normal rat liver using the method of Drysdale et al. (10). The purity of the preparation was checked by SDS-polyacrylamide gel electrophoresis. Specific antiserum was raised in rabbits and purified by affinity chromatography on ferritin-CNDr Sepharose (Pharmacia, Uppsala, Sseden) column. Purified antibodies and normal rabbit immunoglobulins were coupled to peroxidase according to the two step procedure described by Avrameas and Ternynck (11). Sheep anti-rabbit y-globulin antiserum was obtained from>lTInstitut Pasteur, Paris and peroxidase-anti-peroxidase complexes from Miles Yeda Ltd, Rehovot, Israel. fixation-

Tissue

Fixation of liver was performed as previously described (12) with the following modifications: before fixation, rat livers were washed by perfusion via the portal vein with Hepes buffer for 30 sec. then cut into fragments and immersed in 10 % paraformaldehyde buffered with 0.1 M phosphate for 6-8h at 4OC. The samples were washed several times in phosphate buffer and soaked in 10% glycerol in phosphate buffer before freezing in liquid nitrogen-cooled isopentane. Eight-micron thick serial sections were performed for histochemical and immunoperoxidase reactions.

Histochemical

Bile Wachstein

staining

of

canaliculi-membrane and Meisel method

were performed or ATP in the inhibitor.

(13)

for lh at 37’C. incubation medium

Immunolocalization

of

ATPase

activity

bound ATPase activity was revealed by the using sodium ATP as a substrate. Incuba$$ons Controls were obtained with omission of Mg or with 10 mM p-chloromercuric benzoate as an

ferritin

Liver two different

cryostat sections were processed for immunoperoxidase staining by methods a) by the PAP method first using anti-ferritin antibodies then anti-rabbit y-globulin and finally peroxidase anti-peroxidase complexes according to Stenberger et al. (14) ; bl by the direct technique using peroxidase-labelled rabbit anti-ferritin antibodies. Peroxidase activity was demonstrated by the technique of Graham and Karnovsky (15). Control reactions included : a) incubation peroxidase labelled normal antiserum ; b) with c) with normal rabbit serum then with anti-rabbit peroxidase-anti-peroxidase complexes.

with ferritin-absorbed rabbit rabbit immunoglobulins ; y-globulin and finally with

BIOCHEMICAL

Vol. 99, No. 3,198l

The early similar

morpholoqical

to those

vealed

foci

after

wing

nodules.

nique

yielded

At

the

hepatocytes

6 months,

results:

contained

by

both

(4):

PAP

Altered

appeared

and

and

small as gro-

direct

the

in DENA-treated

and

re-

formed

staining

was observed

hepatocytes,

ferritin

no

were

hepatocytes

activity

method

normal

and

COMMUNICATIONS

diethylnitrosamine

by

persisted

the

in

ferritin

RESEARCH

ATPase

lesions

the

ferritin

of

same

by others

in membrane-bound

Immunolocalization all

induced

uescribed

deficiency

3 months.

BIOPHYSICAL

alterations

previousiy

their

by

AND

tech-

rat in

livers,

Kupffer

cells.

However,

in the

consistently

higher

on serial (Fig,

carcinogen-altered than

in the surrounding

all

sections,

normal

ATPase-deficient

1) or in

nodules

(Fig.

Ferritin

and

subunits

hepatocytes.

a strong

staining

been

localized

was

As shown

located

hepatocytes

21 displayed

content either for

foci

in

ferritin.

Discussion

in cultured

and

a diffuse some

rat

A

very

munoperoxidase ment with

cells

(171

fluorescence similar

is

biochemical

if

data

any,

in our in

in

that

showed

“granules”

in

with

the Kupffer

cells

sug-

is

in agree-

cells.

This

the

iron

nodules

in

of

(16)

conditions

these

most

liver

rat

they

fluorescent

of staining ferritin

showing

(18)

with

was observed

The absence

little,

in

by immunofluorescence:

together

pattern

technique. there

that

already

have

hepatoma

cytoplasmic

cells.

gests

its

is

the im-

deposited

as

hemosiderin. The

rat

enhanced

liver

into

the

to store

iron

contrary

an early

observed. the

should

results.

shown).

ferritin thesis.

Secondly

has

into been

could the shown

occur.

completed

shell the

881

these

to

turnover

mechanism a defect

by in

the

the

content

apparently

con-

or an increa-

The latter seems excluded vesicles were seen (results

control

either

induce

in ferritin

On

ferritin

for

in-

hepatocytes content.

in intracellular

endocytotic

lead

to

to iron

reponse

in ferritin

be proposed rate

in the

a modification

in

DENA-treated

of chemically-altered

could could

no

synthesised

increase

a reduced

process

synthesis

subunits

Iron

is

in a decrease

significant

level,

neoplastic

inability

the

explanations

endocytotic

ferritin

and

have resulted

Firstly

the ultrastructural lates

cells,

foci

ferritin

As

and

Several

tradictory se in

of

was unexpected.

corporation

is

staining

which

since not iron

assembly

or to an increase in formation of apoferritin

ferritin from

at

reguof

synfree

BIOCHEMICAL

Vol. 99, No. 3,1981

Figure

AND

BIOPHYSICAL

1 : Light after

microscopy ; serial cryostat sections DENA treatment ; A) AlPase reaction ferritin by the PAP method. The focus of as compared bits a much stronger staining, hepatocytes (x 120).

882

RESEARCH

of

COMMUNICATIONS

rat liver Immunolocalization altered hepatocytes to surrounding

3 months

8)

of exhinormal

Vol. 99, No. 3,198l

Figure

2

: Light after ferritin island some

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

microscopy ; serial cryostat sections of rat liver 6 months DENA treatment ; AJ ATPase reaction U) Immunolocalization by the PAP method. The altered hepatocytes form a larqe which appears as a developping nodule ; note the presence slightly compressed surrounding hepatocytes (arrow) (X 120).

883

of of

Vol.

99, No.

subunits

3,198l

BIOCHEMICAL

dispersed

iron

in

With

the

these

throughout

altered

antibody

staining increase

the

cells

used

corresponds in ferritin

to ferritin synthesis

BIOPHYSICAL

lead study,

or not

to

stimulated

by

COMMUNICATIONS

the

of

discern

subunits

that this finding could be related to the in these hypertrophic altered cells (20). evidence

Therefore

accumulation

we cannot to

RESEARCH

(19).

cytoplasm

could

this

in

AND

unengaged

whether

or whether iron might

of

subunits.

the observed

specific" occur. We suggest

“non

high degree of ploidy which occurs this regard we have preliminary

In

albumin synthesis increases with ploidy by studying separated by counterflow centrifugation in different according to cell size. Whatever is the exact significance that

hepatocytes

tions

absence

normal rat subpopulaof this high

ferritin content of carcinogen-altered hepatocytes, our findings are the first to our knowledge, to indicate that in these cells an intracytoplasmic protein other than an enzyme is modified. Moreover all foci and nodules identified by their deficiency in ATPase activity displayed a higher ferritin Consequently ferritin accumulation could be used as a reliable marcontent. ker

detect

early

Acknowledgements

:

to

lesions

occuring

during

experimental

hepatocarcinogene-

sis.

We acknowledge

nical

We wish to assistance

Pr thank and

M.

Simon for

Mrs A. Saunier and Mrs 0. Houzelle for

This work was supported DGRST

advice.

helpful

by grants

Y. Mazurier typing

the

for their manuscript.

useful

from INSERM (ATP 82-79-114)

tech-

and from

(80-7-6016).

References 1. Merkow, L.P., Epstein, S.M., Caito, B.J., and Bartus, B. (1967) Cancer Res. 27, 1712-1721. 2. Scherer, E., and Emmelot, P. (1976) Cancer Res. 36, 2544-2554. 3. Schmitz-Moorman, Z., Gedigk, P., and Dharamadach, A. (1972) Z. Krebsforsch. 77, Y-16. 4. Farber, t. (1973) in Methods in Cancer Research (Busch, H., ed.) vol. 7, pp 345-375, Academic Press, New York. 5. Bannasch, P., Mayer, D., and Macker, H.3. (1980) Biochim. Biophys. Acta 605, 217-245. 6. Williams, G.M., and Yamamoto, R.S. (1972) 3. Natl. Cancer Inst. 49, 685692. 7. Williams, G.M. (1980) Biochim. Biophys. Acta. 605, 167-189. 8. Fineberg, R.A., and Greenberg, D.M. (1955) 3. ml. Chem. 214, 97-106. 9. Drysdale, 3.W., and Munro, H.N. (1966) 3. Biol. Chem. 241, 3630-3637. 10. Drysdale, J.W., and Munro, H.N. (1965) Biochem. 3. 95, 851-858. 11. Avrameas, S., and Ternynck, T. (1971) Immunochemistry 2, 1175-1179. 12. Guillouzo, A., Belanger, L., Beaumont, C., Valet, J.P., Briggs, R., and Chiu, 3.F. (1978) 3. Histochem. Cytochem. 3, 948-959.

884

BIOCHEMICAL

Vol. 99, No. 3,1981

13. Weichstein, 14. 15. 16.

Stenberger, 3. Histochem. Graham, R.C. 291-302. Lee, I.C.K.,

18.

Lee,

RESEARCH

COMMUNICATIONS

S.S.,

Schlesinger,

(1970) l4,

K.J.,

and

Richter,

G.W.

(1974)

Am.

K.I.,

and

Richter,

G.W.

(1975)

Am.

473-488.

Lee, I.C.K., Lee, S.S., Schlesinger, 3. Pathol. 80, 235-248. Van Wyk, C.P., Linder-Horowitz, M., 246, 1025-1031.

19.

Zshringer, 3., Baliga, Sci. USA 2, 857-861.

B.S.,

20.

Wanson, (1980)

D., Penasse, 459-471.

J.C., Cancer

BIOPHYSICAL

and Meisel, E. (1957) Am. 3. Clin. Path. 27, 13-23. M., L.A., Hardy, P.H. Jr., Cuculis, 3.3., and Meyer, H.G. Cytochem. I& 315-333. Jr., and Karnovsky, M.3. (1966) 3. Histochem. Cytochem.

3. Pathol. 2, 17.

AND

Bernaert, Res. 2,

and

and

Munro,

H.N. W.,

885

Munro,

H.N.

(1971)

Biol.

3.

(1976) Proc. Natl.

Mosselmans,

R.,

and

Chem.

Acad.

Bannasch,

P.