Effect of antioxidants on cultured human diploid fibroblasts exposed to cystine-free medium

Vol.74,

No.4,

1977

BIOCHEMICAL

EFFECT OF ANTIOXIDANTS

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

ON CULTURED HUMAN DIPLOID

EXPOSED TO CYSTINE-FREE Shiro

Bannai,

Hohko Tsukeda,

Division of Biochemistry Tsukuba Ibaraki-ken, 300-31, and Division of Health, Kamiosaki, Shinagawa,

Received

January

FIBROBLASTS

MEDIUM

and Hideo

Okumura

University School of Medicine, of Virology National Institute Tokyo 141, Japan

5,1977

SUMMARY: Logarithmically growing human embryonic diploid cells Glutathione started to die in cystine-free medium within 18 hours. accounted for almost all the acid-solube sulfhydryl compound of the cells and cellular glutathione level decreased rapidly after cystine depletion. By adding vitamin E the cells survived over 6 days in cystine-free medium, though glutathione content of the cells was reduced to less than 1% of the normal level. Synthetic antioxidants had similar effect, and mechanism by which cells die in cystine-free medium was suggested. There

is

requirements

described

culture

(HeLa

diploid

cell

Medium

considerable

due to their that

(BME),

(WI-38)

the utilization rapidly cell

tested

did

resembled of cystine

survival study

the

essential amino

nutrient not

acids

for

All

a vital To better

lines

in Basal and was found

It

in human diploid

(4).

Since

function understand

human

of the ten

(3).

cell

in

for

in a cystine-free

heteroploid

was higher

cell

studied

cysteine

that

nutritional

contained

(l-3).

survive

uptake

was suggested.

the

nutrients

was extensively

acid

medium,

that

two heteroploid

to synthesize

of amino

in cystine-free

following

cystine

inability

closely

for

all

important

lines

the pattern

evidence

Among thirteen

lines.

to be a particularly cell

by Eagle

and L) include

of Eagle

diploid

empirical

medium was also cell

lines diploid

found

lines except cells

of cystine the

human

that died

on the

function,

the

was undertaken. METHODS

Cells used in these experiments were human diploid fibroblast originated in our laboratory from embryonic lung tissue and designated HAIN-6. The culture conditions and subcultivation procedures were

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

1582

I&W

0006-291X

Vol.74,No.4,

1977

BIOCHEMICAL

IIays

after

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

medium

change

Figure 1: Growth profile of HAIN- at various concentrat;on of cystine. Cells were seeded at about 1.2 x lo5 per 30 mn plastic dish with normal BME containing 10% new born calf serum and after a day the medium was changed. Cystine concentration of the changed medium was: 0, two-fold of BME; 0, one-fold of BME (control); A, one-half of BME;X , one-tenth of BME; l , none.

exactly the same as described by Hayflick and Moorhead (5) except that antibiotics were omitted. HAIN- cells were capable of 50 + 5 population doublings and they were studied here at their 16 to 26 passages. For each experiment cells were seeded at the required cell density in Lux plastic dishes and were incubated at 37" in a humid atmosphere of 5% CO -air mixture. Amount of cystine in the serum used for cystinefree a,edium was determined by amino acid analyser, and no free cystine was found. Protein-bound half-cystine residues were measured after treatment of the serum by heat or a reducing reagent, and its concentration was found to be less than 4 nmoles per ml of the serum. Possible effect of contaminating cystine provided by the necessary presence of serum protein in the medium was neglected. Cells were counted with a hemocytometer after treatment of the cells with a solution of trypsin (0.05%) and EDTA (0.5mM). 0.2% nigrosin solution was used as viability stain, when necessary. Cellular acidsoluble sulfhydryls were assayed with 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB) (6). Cellular glutathione was measured by enzymatic method, which is based on the catalytic action of glutathione in the reduction of DTNB by a mixture of NADPH and yeast glutathione reductase (7).

1583

Vol. 74, No. 4, 1977

BIOCHEMICAL

AND BIOPHYSICALRESEARCH

COMMUNICATIONS

, 30 .

.f OJ ;;L “,

20. E

\ I M 0 E ‘0, c

0

L 1

2

IIours

after

3

4

medium

5 change

Figure 2: Changes in contents of acid-soluble sulfhydryls of HAINexposed to cystine-free medium. The dishes containing about 1.5 x lo6 cells were rapidly washed three times with phosphate-buffered saline, and 0.3 ml of ice-cold 15% sulfosalicylic acid was directly added into the dish with 0.6 ml of phosphate-buffered saline. The acid extracts were brought to pH 6.8 by adding 1.2 ml of 0.5 M phosphate buffer, pH 6.8, with 0.3 ml of 1 N NaOH. This was followed immediately by the addition of 0.1 ml of 10 mM DTNB solution. Rapid mixing completed the reaction within a few minutes and the resulting color was read at 412 nm. The points on the graph represent the means of triplicate determinations. 0 , cells treated with normal medium; l , cells treated with cystine-free medium.

RESULTS The effect

of cystine

human embryonic cystine-free

diploid

cells

cystine-depletion,

gated

each other,

were

cells

were

In the

of one-tenth

In the

normal

or one-half

observed presence

BME, the

cells

BME.

observed

with

of normal

of cystine were Since

growing cystine

which

at the in the in the

were

two-fold

1584

at about

final

grew

In 12 hours

and aggremost

of the

concentration at similar

rate

at day 3 or 6, respectively. concentration

same manner culture

1.

rounded

at the

BME, the cells

of the

Fig.

At 30 hours

of cystine

BME, and died

in

started

at 18 hours.

presence

normal

cells,

proliferation

was shown

of the cells

and dead

dead.

on the

in culture

medium degeneration

after

to that

concentration

of the

as was observed

medium was considered

normal with a source

Vol. 74, No. 4, 1977

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Table Amount

Treatment

1

of Glutathione

of HAIN-

Cell

Line

nmole/mg

cell

protein

of Cells Acid-soluble Sulfhydryls

None

32.5

Oxidized Glutathione

Total Glutathione

52.1

33.0

+ 2.5

Cystine-free

Medium

for

6 h

c3.0

1.5 +- 0.4

Cystine-free

Medium

for

24 h*

c3.0

co.2

Vitamin E supplemented free Medium for 6 h

Cystine-

c3.0

1.1 LO.4

Vitamin E supplemented free Medium for 24 h

Cystine-

c3.0

co.2

0.4 -•t 0.2 co.2

co.2

The dishes containing about 1.5 x lo6 cells were rapidly washed three times with phosphate-buffered saline and 1.2 ml of ice-cold 5% trichloroacetic acid was added into the dish. The supernatants were extracted 5 times with 2 volumes of 0.01 N HCl-saturated ether and the aqueous extracts were mixed with 1.2 ml of 0.2 M phosphate-10 mM EDTA, pH 7.0. 1 mol of DTNB, 1.2 unit of yeast glutathione reductase , and 0.2 mol of NADPH were then added to the mixture and absorbancy at 412 nm was recorded. The rate of reaction was usually expressed as the change in absorbancy per 5 min, and the standard curve was run every time. To measure oxidized glutathione cells were pre-treated with N-ethylmaleimide, and excess Nethylmaleimide was removed together with trichloroacetic acid by 10 times extraction with ether. Each value is the mean 5 SD of triplicate determinations. * A part

of cells

of cellular

sulfhydryl

of the cells the

level

level.

In order

predominant

results

total

sulfhydryls

to see what

in Table

it

kind

in the cells,

shown

acid-soluble

medium were

and at 5 hours

medium or of control were

condition.

compounds,

of acid-soluble depletion

free

in this

in cystine-free

cystine

is

died

measured. cells

was less

than

of acid-soluble

were

1, and it

contents determined was evident

1585

contents

As shown in

of the

glutathione cells

sulfhydryl

decreased one-tenth

rapidly

after

compound cells

by enzymatic that

2,

of the normal

sulfhydryl of the

Fig.

amount

in cystinemethod. of total

The

Vol. 74, No. 4, 1977

BIOCHEMICAL

0

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

2 Days

4

after

medium

s

change

Figure 3: Effect of vitamin E on growth of HAINin cystine-free medium. Experimental conditions were the same as described in Figure 1. Vitamin E was used immediately after homogenized in the medium with a sonicator. 0 , control cultures in normal medium; A, cultures in normal medium supplemented with vitamin E (100 ug/ml); *, cultures in cystine-free medium supplemented with vitamin E (100 pg/ml); l , cultures in cystinefree medium.

glutathione

and of acid-soluble

were

equal

and that

it

was concluded

nearly

small. the

Thus bulk

cells

of cellular

exposed There

to remove pherol medium,

is

ester,

had the

that

the cell derivative same effect

ml of the medium,

reduced

glutathione

sulfhydryl

groups.

evidence

that

radicals.

the

cells

number

did

over

not

increase

and synthetic

cells

(GSH) comprised GSH content

in many tissues

in Fig.

6 days during

3, OL-a-taco-

in cystine-free the

DL-a-tocopherol at the concentration

antioxidants,

1586

of the

reduced.

As shown

alive

culture

was negligiblly

GSH functions

of the vitamin, on the

in normal

glutathione

medium was greatly

and free

E) kept

of the cells

of oxidized

acid-soluble

considerable

(vitamin

water-soluble

amount

to cystine-free

peroxides

though

sulfhydryls

butylated

period.

phosphoric

A acid

of 1 ug per hydroxyanisole

Vol.74,No.

4, 1977

and n-propyl

BIOCHEMKAL

gallate,

had also

the similar

of 2.5 ug per ml of the medium antioxidants

was immediate,

one of the antioxidants medium.

In cystine-free

appeared

to be larger

vessels.

Apparently

changed these

to the

cellular

one.

Since

the

GSH contents

had no influence

to the

depletion

vitamin

E supplement

content

as that

1).

for

several

of control

here

However

cultured

(data

on

correlation vitamin

addition

in normal

E on

of vitamin caused

E by

medium with

doublings

not

medium,

had no effect

GSH content

population

cells

in normal

of added

of cellular

Cells

of culture

functional

effect

cells

the medium was re-

that,

employed

a possible

antioxidant,

after

be noted

was investigated.

(Table

the

of

in cystine-free

substratum

indicated

upon the decrease

cystine

with

of these

by addition

to die

was observed

results

concentration

rescued

started

at the concentrations

COMMUNICATIONS

The effect

were

supplemented

It should

GSH and antioxidants,

cellular

cells they

growth

at the

shown).

and more adhesive

normal

growth.

between

is,

medium

effect

not

before

normal

antioxidants

(data

that

just

AND BIOPHYSICALRESEARCH

had the

the

same GSH

shown).

DISCUSSION As is usually almost

all

formation

the

case in mammalian

the non-protein from

of the cells

thiol

cok.metric exposed

to cystine-free

of GSH in embryonic

an hour.

This

kidney

and liver

ported

in skin

may be probable,

fibroblast,

greater)

it

still

a mystery

Depletion

than

(8).

studied

half-life

embryonic

cells

postnatally

here.

sulfhydryls

cells

was approximately to those

found

turnover

rate

of about

6 hours

over

skin

that

in

had been re(9).

grow much more rapidly

in the medium caused

1587

Thus in-

2) suggested

obtained

turned

for

(Fig.

was comparable

Much lower

how GSH is

of cystine

medium

rate

GSH accounted

of acid-soluble

human diploid

with

because

times

is

turnover

--in vivo

three

of the cells

measurements

half-life

high

t issues,

fibroblasts,

This (about though

so fast. acute

decrease

of GSH

Vol.74,No.4,1977

in the

cells

an important by highly

BIOCHEMICAL

followed role

in the

reactive

in cystine-free

by their cell

low GSH content.

E in the

regulation

antioxidants

despite

of their

the view

that

ated

reactions,

nism

involving

cystine-free the

investigation

of the

were

which, GSH. medium

(less

in cystine-free in normal

It

is

provides

of this

also

a suitable

no

studied

here,

Thus the

result

that

in cystine-free

functions

medium

level)

human diploid

experimental

possible

of vitamin

cells

1% of normal

that

because

(12),

may be neutralized

suggested

cells

a role

medium due to free

culture

and other

ll),

was suggested

survived than

damage caused (10,

animal

effective. cells

GSH has

to the damage

in the cultured

antioxidant

die

reduction

metabolism

also

Since

oxidative

in a whole

found

low GSH content cells

against

be much susceptible

of glutathione were

and death.

of oxygen

Although

such interrelationships

in the presence

defence

should

of their

and synthetic

degeneration

intermediates medium

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

model

supports

radical-mediby a mechacells

in

system

for

of GSH.

REFERENCES

:: 3. 4. 5. 6. L: 9. 10. 11. 12.

Eagle, H. (1955) J. Exptl. Med., 102, 37-48. Eagle, H. (1959) Science, 130, 432-437. C., and Friedman, S. M. (1966) Proc. Natl. Eagle, H., Washington, Acad. Sci. U.S., 56, 156-163. Griffiths, J. B. (1970) J. Cell Sci., 6, 739-749. P. S. (1961) Exptl. Cell Res., 25, Hayflick, L., and Moorhead, 585-621. T. (1969) Exptl. Cell Res., 58, 182-185. Ohara, H., and Terashima, Tietze, F. (1969) Anal. Biochem., 27, 502-522. A. (1974) Proc. Natl. Acad. Sci. U.S., Sekura, R., and Meister, 71, 2969-2972. Schulman, J. D., Schneider, J. A., Bradley, K. H., and Seegmiller, J. E. (1971) Clin. Chim. Acta, 35, 383-388. Flohe, L., and Gunzler, W. A. (1974) Glutathione, pp. 132-145, Georg Thieme, Stuttgart. Hogberg, J., Orrenius, S., and Larson, R. E. (1975) Eur. J. Biochem., 50, 595-602. R. A. (1958) J. Biol. Ryerson, S. J., McMillan, P. J., and Mortensen, Chem., 233, 1172-1174.

1588