The effect of adrenalectomy on zinc thionein levels in rat liver

Vol. 91, No. 3, 1979 December

BIOCHEMICAL

14,1979

THE EFFECT

OF

ADRRNALETCMY Frank

*Division artment Vermillion, Received

AND BIOPHYSICAL

0.

CN iINC

Brady+

THIONRIN

and Paul

C.

RESEARCH COMMUNICATIONS Pages 911-918

LRVRLS

IN RAT LIVER

+

Bunger

of Biochemistry, Physiology, and Pharmacology of Anatomy, University of South Dakota School South Dakota 57069, USA

October

and +Depof Medicine,

12, 1979

SUMARY The concentrations of zinc thionein and cytosolic zinc in rat liver were examined in male rats five days after bilateral adrenalectomy . Zinc in netallothionein increased 10 fold, as compared with control animals. Cytosolic zinc increased 79% as compared with controls. 65% of this increase could be accounted for bound to metallothionein. Sham operated animals after five days showed a 4 fold increase in hepatic zinc thionein and a 239Z increase in cytosolic zinc, 71% of this &n&ease being bound to metallothionein. Adrenalectomized rats, maintained on daily injections of corticosterone (4mg/lODg b.w.), exhibited the same levels of zinc thionein and cytosolic zinc as adrenalectomised rats receiving no treatment. Adrenalectomized rats, maintained on daily injections of aldosterone (Sc19/1DOg b.w.), exhibited the same levels of zinc thionein as the sham operated rats, but the cytosolic zinc remained elevated at the level found in adrenalectomised rats receiving no treatment. These results indicate that there is adrenal involvement in the control of hepatic zinc and zinc thionein levels in the rat. Metallothionein eucaryotic mole

is

tissues,

protein.

fication able

which

thionein

which

It

and

in

deal

a 6500

da.lton

binds

has been

molecular

6-7

g-atoms

implicated

zinc

and copper

with

the

weight

protein,

of metal

(Cd,

as functioning

homeostasis.

properties

in

Several

and potential

found

2n,

Cu) per

cadmium

reviews

functions

in

detoxiare

of

avail-

metallo-

(l-3).

Recently,

reports

hepatic

zinc

various

environmental

of

thionein

zinc

cold

environment,

Ccl4

intoxication.

a general inflammation

and

have

zinc

appeared

thionein

are

stresses. in hot

the

livers

of

and

in

infection

have

al.

burn,

increases

levels been

that

was reported

the

to changes (4)

to the strenuous in

the

by

of

exercise, zinc

Stresses

to affect

of

induction

stresses

hepatic

seen.

levels

caused

reported

subjected

heat with

zinc

et

rats

environment,

plasma

indicate

susceptible

Oh,

Concomitant

decline

which

zinc

and thionein,

involving distribu-

0006-291X/79/230911-08$01.00/0 911

Copyright All rights

@ 1979 by Academic Press, Inc. of reprodution in anyform reserved.

Vol. 91, No. 3, 1979

tion

within

BIOCHEMICAL

the rat(5),

changes in hepatic Cousins' zinc,

zinc

has been shown to be correlated

thionein

levels(6).

group(7-10)

has studied

monolayer culture.

or glucagon stimulated a 93% increase

the effects

Stimulation cultures

zinc concmntrations

of zinc levels,

adrenalectomy of zinc

in

male

had no effect

Daily

synthesis prednisolone

rats

in zinc uptake, in copper up-

on zinc uptake

to decrease weight

weanling

effects

liver

and without

thionein

on

parenchymal cells

and a 35% increase

(8).

in HeLa cell administration gains and

(12).

of glucocorticoids

on the uptake

and the subsequent changes in zinc

a study was designed to determine

with

and zinc

in rat

(11).

was reported

In view of these reported

thionein

(9),

Aldocterone

has also been reported

and levels

rat liver

by dexamethasone of metallothionein

three weeks, however,

liver

of glucocorticoids

a 48% (7) and a 106% (8) increase

cells.

with

Dexamethasone in the presence of insulin

in cadmium uptake

take (10) by liver

RESEARCH COMMUNICATIONS

and this

cadmium, and copper uptake by isolated

in primary

for

AND BIOPHYSICAL

steroid

in rat

liver

replacement

the effects therapy

of

on the levels

cytosol.

MATRRIALSANDMRTHODS Hale, Sprague-Dawley rats, 350-4509, were obtained from Sasco, Inc., Omaha, Nebraska. The rats were housed in wire bottomed cages in a temperature and light controlled room and were maintained on Purina rat chow and tap water ad libitum. Following adrenalectomy, rats were maintained on 5% glucose in saline instead of tap water. Aldosterone, corticosterone, and TrisaHCl were obtained from Sigma Chemical Co. Liver cytosol was obtained by homogenization of frozen livers in two volumes of 0.02 M TrismHCl, pH 8.6, 0.25 M sucrose and centrifugation at 4O at 40,000 rpm in a Type 50.2 Ti rotor in a Beckman L5-65 centrifuge to obtain the high speed supernatant or cytosol. Zinc was determined by flame atomic absorption spectrometry, using a Perkin Elmer Model 303. Zinc thionein was quantitated using atomic absorption analysis of the eluate obtained from Sephadex G-75 column chromatograThe zinc content of liver cytosol was phy of liver cytosol (13-17). determined by direct atomic absorption analysis of 100 fold diluted samples. Rats were bilaterally adrenalectomized under ether anesthesia. Anesthetized rats were secured in the prone position on a surgical Their backs were shaved and cleaned with 70% ethanol. board. Bilateral incisions were made just below the last rib and lateral to the deep back muscles. The adrenals were located just above the exposed kidneys

912

Vol. 91, No. 3, 1979

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

and removed by pinching off with a curved forceps. After removal of the adrenals the posterior abdominal wall muscle layer was stitched and the skin wound was sealed with stainless steel autoclips. The surface on and around the incision was coated with bacitracin ointment. The entire process required lo-15 minutes. Rats were housed individually and maintained on Purina rat chow and 5% glucose in saline ad libitum before killing by decapitation on day 5. No animals who survived the surgery died during the five days post adrenalectomy. Sham operated animals received sitilar surgical and postsurgical treatment except the adrenals were not removed. Stock animals, maintained on Purina rat chow and tap water ad libitum, served as controls. Two groups of adrenalectomized animals received daily steroid thereither corticosterone (4mg/lOOg b.w.) or aldosterone (5pg/lOO g b. Corticow.) intraperitoneallyon days 1,2,3, and 4 postadrenalectomy. steronewasadministered as a suspension in olive oil, containing 5% ethanol, and aldosterone was administered in saline. All surgical procedures, injections, and killings were done between 1000 and 1200 hours to avoid effects due to circadian changes in endogenous hormones. Serum corticosterone levels were determined using a fluorometric assay Serum aldosterone levels were not determined. (18).

apy,

**t"

Differences test of

between significance

means were (19).

statistically

evaluated

using

Fisher's

RESULTS The

levels

of

creased

significantly

es were

seen

control

animals,

zinc

in

liver

following

following

in

animals

and

zinc

cytosol

increased

liver

groups.

On an absolute

increase

in

the

sham operated Daily

the

as compared contrast zinc

thionein

prisingly,

and

adrenalectomited aldosterone

reaching cytosolic

the

treatment

levels zinc

cytosolic

of levels

animals,

sham operated in

aldosterone

913

these

71.1%

of

same the

thionein

in

group.

significantly

decrease

zinc

levels,

(P
significantly

decreased with

in

while

in

to zinc

receiving

as compared

with

4 fold

adrenalectomized not

in-

increas-

When compared

bound

did or

greater

to controls,

animals

(P
in

thionein

79% respectively,

for

administration

with

even

adrenalectomized

accounted

(P
zinc

increased

as compared

64.7%

thionein

levels

in

23% and

is

zinc

daily

animals,

group

corticosterone

hepatic

10 fold

zinc

1).

metallothionein

basis,

cytosolic

and

(Table

hepatic

sham operated in

sham operation

adrenalectomy

zinc

and in liver

cytosol

In

hepatic

adrenalectomized

animals treated

(P(O.9). adren,alecto-

Sur-

2:

in

in

Upper zinc

LLower zinc

II

"t"

0.001

0.001

left

hand cytosol.

numbers

are

are

0.05

0.01

comparisons

Diffexences

22.26

0.2

between

between

0.001 0.02 0.4

groups

groups

for

for

0.01

0.001 0.9 0.001

the the

+

19.9

67.4

71.1 64.7

Percent of Cytosolic Zinc Increase in Metallothionein

Means,P<

Adx Aldosterone

Between

+ 0.25

+ 1.38

19.62

Cytosoll liver)

Metallothionein

+ 0.69 f 0.50 f 0.59

in

and

12.02 14.82 21.51

Zinc (w/g

Cytosol

Adx + Corticosterone

of

comparisons

0.4

0.2

0.001 0.01 -

0.011 0.001

o.022 0.001

error.

Hepatic

Adx

Significance

standard

f 0.29

f 0.70

-+ 0.09 f 0.42 + 0.71

Metallothioneinl liver)

in

Sham

of

Control

Test

k

right hand numbers metallothionein.

Control Sham Adx Adx + Corticosterone Adx + Aldosterone

Group

Table

mean

as

1 Values

the

2.69

7

presented

5.77

8

are

0.65 2.64 6.79

in (w/g

4 4 6

Zinc

concentrations

Control Sham Operated Adrenalectomized Adrenalectomized + Corticosterone Adrenalectomized + Aldosterone

Zinc

N

1:

Group

Table

BIOCHEMKAL

Vol. 91, No. 3, 1979

nized

animals

remaining

not

instead

who

received

tests

of

is

did

decrease

at

the

Plasma

of

Table

in

The

between

were

zinc

thionein

for

levels,

animals

complete

means

7.4,

shzun operated,

daily

corticosterone

receiving

no treatment

and

aldosterone

treatment

plasma

l.41~

ml.

Daily

water

should

in

set all

of

the

and

*It**

groups

animals levels

5% glucose

any

lrg/lOO

ml reanimals

In adrenalectomited

adrenalectomized

of

7.6

and adrenalectomized

corticosterone

eliminated and

8.8,

treatment.

consumption

glucocorticoids

the

adrenalectomized

(P(O.4).

levels

receiving

of

RESEARCH COMMUNICATIONS

2,

controls,

have

with

as the

differences

corticosterone

spectively,

parallel

treatment

significance in

in

same levels

no steroid

presented

AND BIOPHYSICAL

sodium

in

or

mineralocorticoids

receiving

decreased

saline

glucose in

animals

in

the

to 0.5-l-5

the

effects

daily

drinking

due

various

to lack

groups.

DISCUSSION Adrenalectomy

dramatically

zinc

in

and

thionein

Sham operation

also

adrenalectomy.

15.4-21.6 ether

in

monal

stress

zinc

to

cytosolic

examined

five

days

after

levels,

but

liver,

as was

laboratory

(14,15),

in

which

pg/g

were

attained

in

liver for

surgery,

control

incorporation

currently

of

the

levels

involve

these

isolated

liver

adrenalectomized

and after control

increases

The

anesthesia

reached

when

levels

by surgery

of

this

liver,

the

caused

mobilization from

rat

increases

but after

of

the

into

being

the

days,

entry

of

thionein,

produced pursued

in

seen

livers

of

in

zinc

into during

915

the the

liver, stress medulla.

of

do not

during

return

to

animals.

and its of

under

are

probably

sham operated

adrenal

laboratory.

zinc

zinc

levels

study,

a

studies

removed

These

cytosolic

as does

to cause

cytosolic

surgically

this

as do the

sufficient

of

surgery.

as greatly

in previous

perfusion.

by the this

is

levels

livers

levels

five

zinc

epinephrine,

rat

not

zinc

subsequent

surgery, This

Hor-

may

question

is

Vol. 91, No. 3, 1979

BIOCHEMICAL

The question

of hormonal control

seems to be more complicated. costerone

or aldosterone

cytosolic

zinc

the levels

lowers these levels surgically

treated

maintain

five

rats,

effects

receiving

corti-

levels

of

Corticosterone

surgery.

zinc

from the liver

elevated

thionein,

found in sham operated

animals received

water ; consequently,

of zinc

their

days after

of hepatic

to that

RESEARCH COMMUNICATIONS

of the exit

Adrenalectomized

therapy,

in the liver

does not affect

AND BIOPHYSICAL

but aldosterone animals.

5% glucose in saline

All

the

as drinking

due to sodium or glucose imbalances should

have been minimized. Cur results with

reports

with

corticosterone

on the stimulation

by dexamethasone (7,8), level

taken up extrahepatic used cortisone

ulate

zinc uptake.

ferential

effect

levels a totally It

of zinc

a direct

thionein

appears that

synthesis of hepatic

influence

potency potency

just

does not

of both ether

followed

this

(20),

may account for and zinc in the

is true,

&es

production

916

then the elevated

animals may be caused by

may have an inhibitory

on metallothionein

anes-

by marked in-

in adrenalectomized

as progesterone

dif-

of hydrocortisone,

netallothionein If

for this

of dexamethasone,

influence

seen in sham operated

aldosterone

(7)

while physiological

and release

mechanism than that

levels

which had already

The explanation

on ACTU secretion,

animals in our study.

different

metallothionein

in HeLa cells,

and the lesser

creases in corticosterone

sham operated

at a normal

dexsmethasone has been shown to stim-

to the greater

stress

elevation

livers

hepatocytes

form of the steroid

Perhaps the strong

which is metabolized.

the trsnsitory

natural

did not (11).

may relate

and surgical

this

production

which is not metabolized,

thesia

were with

Recently,

of hydrocortisone

may have started

compared

CM the other hand one of these studies

and found that

metallothionein

levels

as these cells

zinc.

cannot be directly

of zinc uptake in isolated

while our studies

of zinc,

stimulate

therapy

(11).

animals.

influence

on

Whether this

or an indirect

effect

is

Vol. 91, No. 3, 1979

mediated

either

lothionein

the

BIOCHEMICAL

by zinc

remains

questions

uptake

zinc

moval zinc

remain

by the

of signals,

are

return

to normal.

to

a great

deal

control

of

more

availability

of

this

zinc

or

the

binding

capability

of metal-

to be investigated.

The basic of

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

concerning and what

liver,

necessary The

and

the

involvement zinc

is

mechanisms

subsequent

to allow

transitory

uptake

the

quite

release

by the

was in

preparation,

of

zinc

but

must

or

hepatic

thionein

clear,

liver

control

signals,

levels of

of

re-

cytosolic in

the

of

binding

hormonal

be investigated

in

detail. While

peared methasone thionein

in

this

manuscript

which at

least

14 hours

adrenalectomized

rats

6 days

surgery

after

after injection,

another

receiving

a single

showed

as compared

Supported by a research grant (ES 01288) fun& provided by an interagency grant from FOB is a Research Career Development Awardee DHEW.

report dose

a 50% increase to

(21)

ap-

of

dexa-

in

zinc

controls.

from NIEHS, NIH, DHEW with the EPA (EPA-IAG-DS-E772). (ES 00022) of NIBHS, NIH,

REFERENCES

:: 3. 4. 5. 6. 7. 8. 9. 10.

11. 12. 13. 14. 15.

Kojiaa, Y., and KXgi, J.H.R. (1978) Trends Biochem.Sci. 3, 90-93. D.D. (1972) Ann.Rev.Biochem. 41, 91-128. Vallee, B.L., and Ulmer, Friberg, L., Piscator, M., Nordberg, G.F., and KjellstrUm, T. (1974) Cadmium in the environment, 2nd ed., CRC Press, Cleveland. Oh, S.H., Deagen, J.T., Whanger, P.D., and Weswig, P.H. (1978) Amer.J.Physiol. 234, E282-3285. W.R. (1969) Appl.Microbiol. 18, 482-484. Pekarek, R.S., and Beisel, Sobocinski, P.Z., Canterbury, W.J., Mapes, C.A., and Dinterman, R.E. (1978) Amer.J.Physiol. 234, E399-E406. Failla, M.L., and Cousins, R.J. (1978) Biochim.Biophys.Acta 538, 435-444. Failla, ML., and Cousins, R.J. (1978) Biochin.Biophys.Acta 543, 293-304. Failla, ML., Cousins, R.J., and Mascenik, M.J. (1979) Biochim. Biophys.Acta 583, 63-72. Werner, A.L., and Cousins, R.J. (1979) Fed.Proc. 38, Abs. 1711. Karin, M., and Herschman, H.R. (1979) Science 204, 176-177. Turnlund, J., and Margen, S. (1979) J.Nutr. 109, 467-472. Day, F.A., Coles, B.J., and Brady, F.O. (1978) Bioinorg.Chem. 8, 93-105. Panemangalore, M., and Brady, F-0. (1978) J.Biol.Chem. 235, 78987904. Panemangalore, M., and Brady, F.O. (1979) J.Nutr., in press.

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Vol. 91, No. 3, 1979

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMvtUNlCATlONS

16. Winge, D.R., and Rajagopalau, K.V. (1972) Arch.Biochem.Biophys. 153, 755-762. 17. Winge, D.R., Premakumar, R., and Rajagopalan, K.V. (1975) Arch. BiochewBiophys. 170, 242-252. 18. Guillemin, R., Clayton, G.W., Lipscomb, H.S., and Smith, J.D. (1959) J.Lab.Clin.Med. 53, 830-832. 19. Hodgemau, C.D. (1959) CRC Standard Mathematical Tables, CRC Pub. Co., Cleveland, pp. 250-251. 20. Cook, D.M., Kendall, J.W., Greer, M.A., and Kramer, R.M. (1973) Rudocrinology 93, 1019-1024. 21. Etzel, K.R., Shapiro, S.G., and Cousins, R.J. (1979) Biochem.Biophys.Rer.Commuu. 89, 1120-1126.

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