S-Alkylglutathione, methylglyoxal metabolism and cell division

Vol. 38, No. 4,197O

BIOCHEMICAL

S-ALKYLGLUTATHIONE,

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

MKTHYLGLYOKAL METABOLISM AND CELL DIVISION

Egil

Jellum

and Kjell

Elgjo

Institute of Clinical Bioohemistry and Institute of General and Experimental Pathology, Rikshospitalet, University of Oslo, Oslo, Norway Received

December

1, 1969 SUMMARY

The effects of S-alkylglutathione on methylglyoxal metabolism in vitro and on cell division S-hexyl--in vivo have been studied. &Bone inhibited glyoxalase I, but had no effect on a-ketoaldehyde dehydrogenase from various souraes including sheep, rat and mouse livers, and human malignant tumor cells. The alkylglutathione did not inhibit cell division in an --in vivo mouse epidermis test It seems unlikely that S-alkylglutathiones will cause acsystem. cumulation of ketoaldehydes in cells where both pathways for methylglyoxal degradation are in effective operation.

It

has recently

petitive

inhibitors

converts

methylglyoxal

a-ketoaldehydes It

cellular the only

is

in contrast

(3)

Methylglyoxal

inhibitors

with use other

and certain

and has therefore

(3)

575

can be de(4)

contain

of methylglyoxal

independent.

like

glyoxal,

to

a-ketoaldehyde

The

phenyl-

and 2-keto-3-deoxyglucose

been called

(3).

the glyoxalase

NAD+ or NADP+ as cofactor,

a-ketoaldehydes aldehyde

in cells

microorganisms

oxidation

(2).

the glyoxalase

methylglyoxal

, glutathione

glyoxalase

activity of

However,

by which

requires

and other

of methylglyoxal

(1).

the direct

reaction

hydroxypyruvic

as substrate, genase

pathway

catalyses This

acid.

enzyme can also glyoxal,

acid.

that

growth

Mammalian cells

an enzyme which

but

lactic

been suggested

inhibit

system is not

pyruvic

into

are com-

the enzyme system which

(l),

may be used to cause a build-up

and thus

graded.

of glyoxalase

S-alkylglutathiones

are known to possess carcinostatic

has therefore

system

been shown that

(5) dehydro-

Vol. 38, No. 4, 1970

BIOCHEMICAL

Potential cellular

anticancer

methylglyoxal

the ability

not

only

should

of intra-

consequently

the glyoxalase,

investigation

examines

on the a-ketoaldehyde

including

malignant

tumors

but

possess

also

the a-

of S-alkylgluta-

from various

Possible

from man.

in mouse epidermis

effects

dehydrogenase

methylglyoxal

of S-hexylglutathione, division

based on the principle

dehydrogenase.

The present thiones

agents accumulation

to inhibit

ketoaldehyde

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

inhibitory

and mixtures

--in vivo

sources,

were also

effects

thereof

on cell

tested.

MATERIALS AND METHODS S-hexylglutathione athione

was synthesised

(Sigma Chemical

Switzerland)

Co.,

USA) with

in aqueous ethanol

sodium hydroxide.

The product

gave correct

analysis

for

were carried

out with

S-hexylglutathione,

thione,

but

Solutions acidic lase

I

were prepared

(EC 4.4.1.5)

was obtained

rat,

use with

(adenocarcinoma and retroperitoneal

of the

Ltd.,

by Vince

experiments is

almost

compounds (1).

earlier

(5).

from Glyoxa-

and Sohne, (O.OlM,

dehydrogenase

as

gluta-

free

buffer

and from two

Germany,

pH 6.6).

were prepared

malignant

tumors

old male patient,

tumor from a 45 year

576

of

England)

from Boehringer

blendor

excess

and S-octyl

as described

were homogenized

in a Waring

this

the latter

stomach from a 60 year

neurogeneous

The tissues

sodium chloride

than

phosphate

AG.,

The product All

I as S-heptyl

of a-ketoaldehyde

mouse and sheep livers

from water.

glut-

(Fluka

as described

since

(Koch-Light

contaminants

before

a slight

analysis).

more soluble

of methylglyoxal

Crude solutions

patient).

N (Kjeldahl

of glyoxalase

considerably

and was diluted

from

twice

reduced

iodide

was isolated

and recrystallized

an inhibitor

hexyl

containing

and Wadd (1)

potent

by reacting

in 5 volumes

and sonicated

for

old

female

of 0.15 30

M

seconds

Vol. 38, No. 4, 1970

(20,000

BIOCHEMICAL

cycles/set

centrifugation

and 6A) with

at

105,000

were passed through librated

with

endogeneous tides.

from

This

G-25,

equi-

removed all

and pyridine

nucleo-

was used as enzyme.

preparation

by extraction

of acetone

and fractionation (5)

In

dehydrogenase powder with

of glyoxalase

I was measured as increase

at 240 mu and the activity as increase

spectrophotometer

following

the method

(methylglyoxal

The effect was tested

(for

which

proliferation discussion

gether

the substance

fracture

routine

were stained

exactly dehydration

with

represents

during

the 4 hour

hours

epidermis.

This

cells

as assay

in vivo -system.

In all

intraperitoneally

to-

injection

were killed of Colcemid.

the sections

and mitoses

were counted

The number of Coloemid that

has entered

method has been extensively

577

The

6).

embedding

the number of cells period.

(5).

The animals

after

and paraffin

the

the 2,4-

see reference

to be tested. four

using

of

(5).

by means of the Colcemid

was injected

Celestine-Blue/haemalum

in 8 mm interfollicular mitoses

on dividing

procedure

0.15 mg of Colcemid

described

described

was estimated

experiments with

was determined

mouse epidermis

of this

at 340 mu in a Zeiss

as previously

method previously

the hairless

dehydro-

measured as disappearance

of S-hexylglutathione

with

of cell

(RPQ 20A),

),

in the

of the a-ketoaldehyde

in absorbance

were also

dinitrophenylhydrazine

After

supernatants

described

The enzyme activities

by neck

clear

as previously

genase was followed

method

After

(3).

absorbance

substrate

the

a-ketoaldehyde

involved

followed

The activity

recording

fraction

purified

The purification

of Monder

sonifier.

(pR 7.9).

glutathione

protein

a partially

sheep liver,

30 minutes

buffer

including

The gelfiltered

ammonium sulphate

rate

x g for

0.1 M phosphate

some experiments

S-75

a Branson

a column (30 x 13 cm) of Sephadex

cofactors

was used.

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

arrested

mitosis used

Vol. 38, No. 4, 1970

for

testing

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

the activity

of various

substances

The cytostaticum

mitotic

rate

control

in the present

produce

a mitotic

(7).

methotrexate

experiments

depression

on the

since

in the

this

epidermal

was used as a agent

interfollicular

is known to epidermis

(8).

RESULTS Fig.

la

glutathione

confirms inhibits

the finding glyoxalase

GLYOXALASE

of Vince I.

I

and Wadd that

Our data

show that

C~-KETOALDEH.YDE

MINUTES

S-hexylapproximately

D~YDR~GENASE

MINUTES

la. Effect of S-hexylglutathione on glyoxalase I. %-assay mixture contained: phosphate buffer (pH 6.6) (67d) glutathione (1.3mM), methylglyoxal (1.3&4), glyoxalase I (5 pi of protein) and S-hexylglutathione in conoentrations as stated below, in a final volume of 1.5 ml at 220.

I control without hexylglutathione = 0.08 mM hexylglutathione - 0.8 mM hexylglutathione

Fig. lb. Effect of S-hexylglutathione on partially purified sheep liver a-ketoaldehyde dehydrogenase. The assay mixture containeda Tris buffer (pH 7.8) (lOOmM), methylglyoxal (5mM), sheep liver enzyme (40 pg of protein), NAD+ or NADP+ (0.6m~) and S-hexylglutathione (1OmM) in a final volume of 1.6 ml at 220.

-0

-O-

-a

-a-

P control without hexylglutathione = with 10 YIN hexylglutathione 578

Vol. 38, No. 4, 1970

0.04

mM final

BIOCHEMICAL

concentration

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

of S-hexylglutathione

cause 5046 inhibition

of the glyoxalase,

the results

and Wadd (1).

of vince

S-hexylglutathione tide of

large

oxidation

is effect

to pyruvate

and rat

also

had no inhibitory

effect

the

that nucleo-

sheep liver

the oxidation

of methyl-

as in the control

(Fig.

lb).

dehydrogenases

were used (not

shown).

The two

a-ketoaldehyde

dehydrogenase

activity,

density

340 mu as well

as dis-

in optical

of substrate

with

Even in the presence

when a-ketoaldehyde

livers

contained

measured as increase appearance

the same rate

was obtained

from mouse livers tested

at

however,

on the pyridine

(1O’2#)

catalyzed

to

good agreement

evident,

of methylglyoxal.

dehydrogenase

The same result

tumors

It

amounts of S-hexylglutathione

a-ketoaldehyde glyoxal

in very

has no inhibitory

dependent

was sufficient

at

(methylglyoxal). on the tumor

S-hexylglutathione dehydrogenase

activity

(0.01

M)

(Fig.

2).

0.3 .

t 0 \+ n) L ;

0.1

2

4

6

8

10

MINUTES

Fig. 2. Effect of S-hexylglutathione on a-ketoaldehyde dehydrogenase from adenocarcinoma cells. The assay mixture contained: sodium phosphate buffer (pH 7.9) (8OmM), methylglyoxal (5mM), gelfiltered extract of adenocaroinoma cells (200 pg of protein), NAD+ (0.6m~) and S-hexylglutathione (10&f) in a final volume of 1.6 ml at 220.

-O-0

O-a-

= control without hexylglutathione = with 10 mM hexylglutathione

579

Vol. 38, No. 4, 1970

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

TABLE I EFFECT OF S-HEXYLGLUTATHIONE AND METHYLCLYOXAL ON THE EPIDEBMAL MITCTIC BATE

Number of Animals

Treatment

Methylglyoxal (25 )unoles i.p.)*

Number

12

of arrested Mitoses

23.33

-, 1.12

S-hexylglutathione (5 pmoles i.p.)

8

24.62

2 2.87

S-hexylglutathione (5 pmoles i.v.)*

7

23.00

+ 4.04

8

23.50

+ 3.60

17

24.17

+ 1.98

8

15.75

2 2.45

~-~~;,:;t~t~ione Methylglyoxal (25 poles)

i.p.

Controls (no treatment) Controls Methotrexate,

i.p.)**

* Abbreviations:

i.p. = intraperitoneally i.v. = intraveneously Total volume injected into each animal: ** 5 mg/kg body weight

Table division

I shows that

and methylglyoxal

system.

It

injected

into

should

be noted

- 0.7

inhibit

mM which

that

to obtain is about

the glyoxalase

&en bad no

the mice (5 poles

25 g) was sufficient 0.6

S-hexylglutathione

in the mouse epidermis.

glutathione

0.5

ml.

had no effeot a mixture

effect

of

on the oell

the alkylated

in our --in vivo

test

the amount of S-hexylglutathione per mouse weighing a concentration

15 - 20 times

50s.

580

higher

approximately

in the animal than required

of to

Vol. 38, No. 4, 1970

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

DISCUSSION The physiological it

has been suggested

in

the regulation

tie

therefore

remains

substances,

I,

which

therefore

methylglyoxal

the failure

of Monder

and lung

dehydrogenase certain contain exists,

(3)

activity cancer

(3). cells,

a-ketoaldehyde however,

that

(1).

a potent

inhibitor

directly

will

operation.

This

probably

to inhibit is

cell

whether

a-ketoaldehyde

enzyme,

(sheep)

dehydrogenase

cells,

e.g.

kidney

although high

shows that

of an adenocarThe possi-

activity.

of cancer

all

show a very

investigation

such as the cells

division

dehydrogenase.

of the

581

cause for

are devoid

types

(3).

pathways

organs,

The present

the

where both

several

species

of

to pyruvate

that

other

of the

on the a-ketoaldehyde

question

contain

from a different

3).

The present

in cells

indicate

that

to a build-up

S-alkylglutathiones

Another

apparently

suggest

(see reference

has no effect

cells,

in tissues

and Wadd

methylglyoxal

system.

procedures

growth-retard-

being

that

readily

to be powerful

are in effective

cancer

from rat

reports

of S-hexylglutathione

test

and in particular

bility

although

degradation

however,

are found

leading

prove

of ketoaldehydes

in our Ain vivo

cinema,

several

by Vince

unlikely

is,

ketoaldehydea

intermediate

might

converts

appears

an accumulation

least

cells

tissues

ketoaldehydes

S-hexylglutathione

dehydrogenase

and lung

be involved

down growth-retarding

that

metabolism

shows that

The results

although

recommended isolation

but

as suggested

investigation

at

whether

unsettled,

in the

in

the finding

during

of methylglyoxal

explains

by breaking

may be a metabolic

ketoaldehyde

kidney

after

question

methylglyoxal

It

division

as artifacts

(10-12).

glyoxalase

known,

enzyme system might

of a-ketoaldehydes

particularly

are produced

ing

this

is not

(9).

The occurrence

Inhibitors

of glyoxalase

that

of cell

a-ketoaldehydes

doubtful,

role

cells

might

be

Vol. 38, No. 4, 1970

devoid will

of

BlOCHEMlCAt

the enzyme.

S-alkylglutathiones

It

AND BIOPHYSICAL RESEARCH COMMUNKATIONS

appears

likely

show growth

that

retarding

only

in such cases

activity.

ACKNOWLEDGEMENTS The skillful technical assistance of Mrs. Anne Marie Lund and Mrs. Wenche Edgehill is gratefully acknowledged. The research was supported by a grant to E. J. from the Norwegian Cancer Society.

REFERENCES Vince, R. and Wadd, W.B., Biochem. Biophys. Res. COIMI. 2, 593 (1969). 2. French, F.A. and Freedlander, B.L., Cancer Res. 18, 172 (1958). Monder, C., J. Biol. Chem. &, 4603 (1967). 2: Higgins, 1.3. and Turner, J.M., Biochim. Biophys. Acta 181(, 464 (1969). Biophys. Acta 165, 357 (1968). Jellum, E., Biochim. Colchicine in Agriculture, Eigsti, O.J. and Dustin, P., jr., Medicine, Biology, and Chemistry. The Iowa College Press, Ames (1955). W.S., Laurence, E.B., Iversen, O.H. and Elgjo, K., 7. Bullough, Nature (Lond.) 214, 578 (1967). Robinson, T.E.W. and Stoughton, R.B., J. Invest. Dermatol. 2, 8. 223 (1969). A., Bioelectronics, Academic 9. Saent-Gyorgyi, 10. Riddle, V. and Lorenz, F.W., J. Biol. Chem. 11. Jellum, E., Biochim. Biophys. Acta a, 430 12. Otsuka, I-I. and Egyud, L.C., Biochim. Biophys. Acta 165, 172 (1968). 1.

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