Vol. 79, No. 1, 1977

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

SELENIUM INDUCED GLUTATHIONE PEROXIDASE ACTIVITY IN MOUSE NEUROBLASTOMA CELLS G. S. Germain Department Sciences,

of Biochemistry, Memphis, TN 38163

Received

September

and R. M. Arneson

University

of Tennessee

Center

for

the Health

27,1977 Sumnary

Mouse neuroblastoma cells grown in medium containing 10 percent fetal bovine serum have negligible amounts of glutathione peroxidase activity. Introduction of selenite to the medium to produce a concentration of 600 nM resulted in a 30-fold increase in the enzyme activity. This increase is directly proportional to the concentration below 60 nM and levels Off at concentrations above this value. Selenate produces no increase in enzyme activity when present alone nor does it inhibit induction when present with selenite. Tellurite produces no increase in enzyme activity when present alone but does inhibit induction when present with selenite.

Selenium culture. WI-38

has been shown to be an important

McKeehan cells

--et al.

in tissue

(1)

demonstrated

cultures

with

added

serum requirement

for

from

bone marrow

could

by Se.

properties

selenoenzyme

glutathione

doreductase,

EC 1.11.1.9)

dant

capacity

activity

of Se may result (GHS) peroxidase (3).

to the concentration

that

the

initially

formation

its

speculate

we assayed

low enzyme activity

size

(2)

cells the in the

peroxide of the

their

of

found

that

incorporation

of an investigation

cells,

colony

by haemopoietic

Both groups from

in tissue

--et al.

(glutathione:hydrogen

As part

of mouse neuroblastoma

and found

colony

cells

in the

Se and Guilbert

some of the

growth-promoting

for

an increase

that

be replaced

nutrient

oxi-

antioxi-

GSH peroxidase was very

sensitive

of Se in the medium. Materials

and Methods

Mouse neuroblastoma cells (clone N18) were grown in Dulbecco's modified Eagle's medium (4) without antibiotics or antimycotics (KB Biological, Inc.) supplemented with 10% fetal bovine serum in a humidified atmosphere of 10% CO2-90% air at 37°C in Falcon 75 cm2 flasks. Supplementation of the media was achieved by adding appropriate aliquots of 150x stock solutions of neutralized selenious acid ("specpure" grade from Johnson Matthey Chemicals Ltd.), sodium selenate (Pfaltz & Bauer Inc., Stamford, CT) and sodium tellurite (Pfaltz & Bauer Inc., Stamford, CT) to each selected flask. Control Copyright All rights

0 1977 by Academic Press, Inc. of reproduction in any form reserved.

119 ISSN

0006-291X

Vol. 79, No. 1, 1977

Table

I

Days in Culture 2 4 6

BIOCHEMICAL

Effect of Selenite, Peroxidase Activity

AND BIOPHYSICAL

Selenate

Treatment

RESEARCH COMMUNICATIONS

and Tellurite

on Glutathione

Specific Activity Units/mga

C Serum (A)b E; Serum (A) + 30 nM Se Serum (A) 0:21 Serum (A) + 30 nM Se 9.1 Serum (A) 1.15 + 0.586 Dialyzed Serum (A) 0761 Serum (A) + 15 nM Se 6.6 Serum (A) + 30 nM Se 13.8 + 1.6 Serum (A) + 60 nM Se 26.5 T 7.2 Serum (A) + 300 nM Se 33:6 Serum (A) + 600 nM Se 34.6 Serum (A) + 6 uM Se 29.4 Serum (A) t 60 pM Se 29.3 No Serume N.D. No Serum + 30 nM Se 14.0 Serum (B) 1.4 Serum (B) + 30 nM Se 15.1 Serum (C) 2.8 Serum (C) + 30 nM Se 20.0 Serum (A) + 30 nM Se VIf 1.46 Serum (A) f 60 nM Se VI 1.72 Serum (A) + 30 nM Se + 30 nM Se VI 13.7 Serum (A) + 30 nM Tef 0.70 Serum (A) + 30 nM Se t 30 nM Te 5.8 Serum (A) + 30 nM Se t 60 nM Te 2.4

aEnzyme units are expressed as nanomoles of NADPH oxidized per minute at 24°C. The non-enzymatic rate has been subtracted. The average non-enzymatic rate is 0.52 nanomoles of NADPH oxidized per minute. bGrowth medium consists of 10% v/v fetal bovine serum plus 90% v/v Delbecco's modified Eagle's medium. The letter in parentheses indicates the particular serum lot. CNot detectably different from corresponding non-enzymatic rate. dstandard deviations are only given for those enzyme activities that were determined by three or more experiments each on a different passage. The values with no standard deviations are the average of two experiments except those of serums B and C which are the averages of three determinations done on one experiment. eThe cells in this experiment were fed serum-supplemented medium for four days and serum-free medium for the final two days. fSe VI represents represents selenium introduced as the selenate ion. Te represents tellurium introduced as the tellurite ion.

flasks received equivalent volumes of the diluent. Each treatment or control group always contained four flasks and was considered a single experiment. Flasks were fed every other day at which time either selenite, selenate, tellurite or diluent was also replenished. Cytodifferentiation of neuroblastoma cells was initiated by removal of serum from the culture medium (5) and these cells were then fed every day until they were harvested.

120

BIOCHEMICAL

Vol. 79, No. 1, 1977

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

The cells were harvested by discarding the growth medium and removing the The harvested cells from four flasks cells with an EDTA solution pH 7.2 (6). were pooled, centrifuged and the supernatant discarded. The pellet was resuspended and homogenized in 25 mM potassium phosphate buffer pH 7.0, containwas carried out using a polytron ing 100 mM KC1 and 1 mM EDTA. Homogenization (Brinkmann Instruments) operated at an intermediate speed for two thirty secThe homogenate was immediately centrifuged at 112,000 X g ond periods at 0°C. A sample of the supernatant was removed for a protein deterfor 60 minutes. mination (7) and the remaining supernatant was placed in a plastic pro-vial Within three days the vials were (Costar) and frozen in liquid nitrogen. thawed and assayed by a modification of the method of Paglia and Valentine (8) using cumene hydroperoxide dissolved in 95% ethanol as the substrate (9, 10). Supernatants stored in liquid nitrogen did not lose their activities after fifteen days when compared to the same supernatants assayed fresh, immediately after isolation. Cumene hydroperoxide (Pfaltz & Bauer Inc., Stamford, CT) was assayed for purity (11) and found to have a 81.5% peroxide content. The final concentrations of the reagents in each cuvette were as follows: 91 mM potassium phosphate pH 7.0, 3.6 mM EDTA, 2 mM GSH, 0.1 mM NADPH, 0.16 mM cumene hydroperoxide, 1 unit of glutathione reductase (Sigma) and a 0.1 ml sample of the supernatant or in blank cuvettes 0.1 ml of homogenization buffer. A 100 ml sample of serum lot A was dialyzed for 24 hours against 2 liters of 0.13 M sodium chloride. After 24 hours the saline was changed and the dialysis was continued for 26 hours. The dialyzed serum was then sterilized by filtration through a type HA Millipore filter.

GSH peroxidase was uniformly

it

produced

both

activity

low.

GSH peroxidase

Addition

activity. optimal

(Table

activity

This

particular

in the

experiments

of GSH peroxidase

unsupplemented

in a dramatic

Se concentration

and unsupplemented

there

the longer

the

increase

was chosen

of McKeehan

media

with

--et al.

cells

were

in because

(1).

was an increase

Se

With in the

in culture

I). The anomalously

and unsupplemented

low enzyme activity two-day

cells

The low enzyme content

vested. only

slightly

both

the blank

negative

greater

than

for

in these

values

resulted of the

resulting

and the enzyme activities

two-day averages

can occur. cells. were

vary

low high

individual

same magnitude

Se supplemented

number speed

independently

The resulting

121

for

non-enzymatic

Some of the of the

obtained

from the

the corresponding

enzyme activities

much lower cluded

of 30 nM Se resulted

growth

Se supplemented

specific

Results and Discussion of cells cultured in media

average

of cells supernatants

blank.

each assay, therefore

enzyme activities as those

was

Since

within is

har-

of four-day

in-

Vol. 79, No. 1, 1977

cells.

BIOCHEMICAL

When cultured

six

and undifferentiated

cells

Neuroblastoma concentration 60 nM. cific low

levels

GSH peroxidase

alyzed trol

cultures. there

tracted.

This

cell

number,

selenate

experiments

or tellurite

cell

dropped

the

here

media

was equalled

pellets

were

red

the

at a concentration Selenate dase activity. selenite. simultaneously Similarly,

of selenite.

112,000

x g centrifugation.

Apparently Selenate

also

present tellurite

mitosis

cell

does not

con-

in serum no readily

exceed

6 days,

during

which

not

differ

is ex-

the

media

from

the cultures

red

The inference

has no mechanism inhibit

Viability

al-

containing sele-

remained

with

of selenite

(12).

to reduce

GSH peroxidase

concentra-

of elemental

selenium

has been noted

a

concentrations

number.

presence

This

until

At this

two higher

control

time

among selenite,

of 30 nM and 60 nM had no effect the

for

but

by the

reduction

at concentrations

on di-

is active

of the

colored

of 10 uM with

grown

which

and for

and the cell

after

from

no detect-

compound

or exceeded.

drastically

pellet

The

enzyme activity

and unsupplemented

so decreased

the

spe-

Se supplementa-

Cultures

normal

did

and one-tenth

by the

but

(1).

to one-fifth

nium produced

without

some selenium

was reduced

30 uM and 60 uM selenite

of

concentrations.

two days.

content

by one-half

increase

when serum was removed

after

described

supplemented

to the

and no Se was added,

by others

of 6 uM selenite number

grown

compound

and protein

activity.

a concentration

a small

by cells

while

a selenium

below

higher

serum for

that

specific proportional

all

one-half

indicates

viability

concentration tion

about

has been noted

None of the

produced for

remained

only

remains

ion

differentiation

activity

This

removable

value

of Se in the

serum produced

selenite

exhibited

to achieve

of 30 nM Se, differentiated

is directly

constant

of GSH peroxidase

RESEARCH COMMUNICATIONS

same GSH peroxidase

as the

was essentially

cultures

presence

activity

above this

are due to traces

able

had the

GSH peroxidase

Concentrations

control

in the

of Se introduced

activity

tion

days

AND BIOPHYSICAL

activity

on GSH peroxithis

form induced

to by

selenite. produces

no increase

122

in GSH peroxidase

activity

when

BIOCHEMICAL

Vol. 79, No. 1, 1977

present

alone.

Oppositely,

peroxidase

activity

enzymatic

glutathione

hibiting

oxidation present

point

influence

in the is

the enzyme resulting pending

does

proportionally

known to be produced

by simultaneously At what

it

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

by selenite

tellurite

(13).

induction

of GSH peroxidase

Tellurite

in hybrid

forms

number

the

by 30 nM selenite

is catalyzed

unknown.

on the relative

decrease

present

tellurite

and tellurium

the selenate

and tellurite

Non-

inhibited

exerts

its

in-

be incorporated

have no or different

of selenium

GSH

alone.

and is also

may in some form

which

expected

into

activities

atoms

de-

in each enzyme

molecule.

In any case the data the

idea

that

from

the oxidation

state

of the metal

Such a system of neuroblastoma as an excellent

model

novo or pre-existing susceptibility levels

molecules

for

Se, particularly

these

possibilities research

are

cells

GSH peroxidase

the amounts being

was supported

by the

to lipid

Pragmatically,

be determined.

assay

whether

is of great

is

presence

available

serve

synthesized

-de

Also

at various

could

in serum

serum would

of Se.

peroxidation the system

support

importance.

and a Se depleted

are activated

of neuroblastoma

could

This

to determine

cells

experiments

the

Se

be used as a bio-

samples.

All

of

explored. by National

Cancer

Institute

Grant

CA 16927.

REFERENCES McKeehan, W.L., Hamilton, W.G., and Ham, R.G. (1976) --Proc. Natl. g. Sci. U.S.A. 73, 2023-2027. -and Iscove, N.N. (1976) Nature 263, 594-595. Gullbert, L.J., 32: Hoekstra, W.G. (1975) 7--Fed. Proc. Am. Soc.Exptl.iol. 34, 2083-2089. 4. Dulbecco, R. (1970) In Vitro 6, 5. 5. Seeds, N.W., Gilman, A.G., Anamo, T., and Nirenberg, M.W. (1970) m. Natl. Acad. Sci. U.S.A. 66, 160-167. 6. Paul, ml9m Cell andTissue Culture, p. 218, E. & S. Livingston, Edinburgh‘and London. 7. Lowry, O.H., Rosebrough, N.F., Farr, A.L., and Randall, R.J. (1951) 2. Biol. Chem. 193, 265-275. 8. mia, D.E., and Valentine, W. N. (1967) J. Lab. Clin. Med. 70, 158-169. 9. Little, C., Olinescu, R., Reid, K.G., and-O'Brien, P. J .797m J. m. Chem. 245, 3632-3636. Prohaska, J.R., and Ganther, H. E. (1976) J. Neurochem. 27, 1379-1387. Z: A.J. (1964) AnaT. e. 36, 194-204. Mair, R.D., and Graupner, 12. Hughes, A.F.W. (1950) Quart. J. Microsco ) Tg. 9l-, 251-276. 13. A.L. n958 J. Biol. Chem. 233, 1230-1232. Tsen, C.C., and Tappel,

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