Binding of ethacrynic acid to hepatic glutathione S -transferases in vivo in the rat

Biochemical Pharmacology, Vol. 29, pp.1205.1208. Pergamon Press Ltd. 1980. Printed in Great Britain.

BINDING OF ETHACRYNICACID TO HEPATIC g-TRANSFERASES

Tadataka

7 January

acid is a substrate

their study defining olism and biliary but consistent

and Research

1980; accepted

have undertaken

Such an association

these studies,

to the glutathione

would

therefore,

to characterize

& Dohme (Rahway,

from the Sigma

Corp.

Chemical

other chemicals us:din Preparation

were anesthetized

placed on a heated operating

sp. act. 2 $i/mg) the animals

livers were removed crose buffer homogenates struments,

M sodium quots

phosphate

were chromatographed phosphate

buffer

Boston,

scintillation

spectrometer

GSH conjugation containing

intravenously.

Chemical

of each fraction

and

["Clethacrynic

the abdominal

(v/w) 0.1 M sodium

acid

drug adminis-

aorta.

phosphate,

The 0.25 M su-

tissue homogenizer.

TJ-6

centrifuge

The supernatant

against

The

(Beckman

fractions

L3-50 ultracentrifuge

(2

In-

were

at 4" and the

three changes of Sixty ml of the di-

(5 x 100 cm) at 4" using a 0.01

, and 10.2ml fractions were collected.

to vials containing was measured

with quench correction

the bulk of enzyme activity

300 g,

(50 mg/kg)

Ninety min following

through

G-100 column

were added

All

WI).

products.

(pH 7.4) over 36 hr at 4O.

MA), and radioactivity

activity

and

were pooled and dialyzed

(pH 7.4) for elution

(CDNB)

approximately

of pentobarbital

at 4" in a Beckman

buffer

was ob-

(GSH) was ob-

Co. (Milwaukee,

commerical

pestle and a motor-driven

on a Sephadex

(0.1 ml) of the fractions

land Nuclear,

Glutathione

IL).

were opened and their renal pedicles

for 60 min in a Beckman

thus obtained

acid

16 pCi/mg) were

(49 $i/nunole)

rats, weighing

CA) at 1OOOg for 15 min.

at 100,OOOg

We

of ethacrynic

[2-'%j,

veins were cannulated,

centrifuged

bind-

of this binding.

I-chloro-2,4_dinitrobenzene

injections

with 2 parts

(pH 7.4) using a Teflon

fractions

Heights,

Their abdomens

jugular

and homogenized

1000 ml of 0.01 M sodium alysate

intraperitoneal

was injected

Inc., Fullerton,

supernatant

(Arlington

were killed by exsanguination

were immediately

ultracentrifuged

(phenoxyacetic ["ClMethyl-iodide

NJ).

(DCNB) from the Aldrich

board.

Their left external

to

of covalent

AND METHODS

acid

Six male Sprague-Dawley

with single

and covalently

the binding

this study were readily available

of tissues.

that a small

enzyme activation.

the selectivity

Co. (St. Louis,MO)and

and 1,2-dichloro-4-nitrobenzene

In

in the hepatic metab-

a unique example

prior microsomal

and to delineate

acid and labeled ethacrynic

tained from the Amersham/Searle

tration,

--in vitro (1,2).

$trans.ferases

represent

enzyme without

g-transferases

gifts of Merck, Sharpe

mg/kg,

1980)

acid, Wallin -et al. (3) suggested

MATERIALS

ligated.

CA, U.S.A.

portion of the adniinistered drug was bound selectively

ing of a drug to the metabolizing

tained

Los Angeles,

z-transferases

of the glutathione

of ethacrynic

VA Wadsworth

5 February

for rat glutathione

the importance

excretion

the transferases.

Ethacrynic

Services,

Center, and UCLA School of Medicine, (Received

Ethacrynic

IN VIVO IN THE RAT --

Yamada and Neil Kaplowitz

Gastroenterology Medical

GLUTATHIONE

1205

II

LS-3150T

using external, standardization.

was measured

(fractions

10 ml Aquasol

in a Beckman

(see below),

Ali-

(New Engliquid CDNB-

and the fractions

82-96) were pooled.

Aliquots

of the

1206

Preliminary Communications

pool were assayed lent binding

for radioactivity,

(see below).

of 0.01 M Tris-Cl

protein

concentration

The rest was dialyzed

(pH 8.0), and batch adsorbed

with the same buffer.

at 4" against

swollen

ing

to the method of Habig -et al. (2), by dialysis

column

phosphate

buffer

(I.5 x 40 cm), and elution

same buffer.

The obtained

iodide conjugating Enzyme assays, of Booth --* et al

The enzymatic

sample at 370.

5%

ethanol,

The DCNB-GSH

(5).

The reaction

between

were assayed

reaction

formation

buffer

GSH and methyl-iodide

taken at 0, 1, 3 and 5 min, transferred

Hot distilled

water

(1.0 ml) was added,

ty of the non-volatile the reaction

was linear

Measurement eluates

at 3000 r.p.m.

radioactivity

binding.

quots of 50% trichloroacetic tected

in the supernatant washes

were dissolved assayed order

by 10 ml

spontaneous

until no further

background

(0.5 ml) were vials containheating.

II, and the radioactiviUnder these conditions, interaction

of substrates.

(2 ml each) of the pool of Sephadex

fractions

Then the samples

decanted

and assayed

radioactivity

and counted

for

could be de-

in a similar

The remaining

fashion

precipitates

at 37" for 15 min, and 200 ~1 aliquots

readings.

G-100

acid, cen-

and washed with 0.5 ml ali-

were treated

and 2 ml chloroform.

in

or ab-

hood with gentle

Aquasol

acid repeatedly

Blanks were prepared

of

was incubated

with 0.5 ml of 50% trichloroacetic

in 1 ml of 1 N NaOH by heating

incubation

incubation

Aliquots

as described.

were suspended

fractions.

appropriate

(6,7).

The precipitates

using 2 ml methanol

for radioactivity.

to obtain

nm following

to liquid scintillation

significant

Six aliquots

using the method

(pH 8.0) and 50 ~1 of sample at 37"

for 2 min, and the supernatant

as described.

with repeated

followed

enzyme were precipitated

and GSH-methyl-

at 340 nm following

under a ventilated

product was measured

for 5 min without

of covalent

containing

trifuged

reaction

GSH-DCNB

(pH 7.0) at 25" in the presence

immediately

and then blown to dryness

(CM-52)

(pH 6.5) and 1 mM GSH with 10 ~1 of

sence of 200 ~1 of the test sample as the source of enzyme

ing 1.0 ml methanol,

of 2000

of 0 to 2011 mM KC1 in the

(1.7 mM, 0.02 uCi/umole)

2 mM EDTA buffer

DE-

accord-

and conductivity.

was also followedat phosphate

three changes

CDNB and GSH was assayed

phosphate

(Whatman

purified

on a CM cellulose

for GSH-CDNB,

product

0.1 M sodium

reaction

phosphate,

at 4" against

radioactivity,

between

reaction

0.1 mM CDNB, 10 mM GSH, 0.1 M sodium

3 ml of 66 mM sodium

was further

(pH 6.7), chromatography

(see below),

(5) by measuring

thus obtained

with a 100 ml linear gradient

fractions

activities

of 0.01 mM CDNB in

The eluate

acid cova-

three changes of 2000 ml

with 100 ml of DEAE cellulose

52)

ml of 0.01 M potassium

(4), and ethacrynic

for each set of samples

Quench correction

was by external

were in stan-

dardization. RESULTS As depicted sol eluated

Fig. 1.

in Fig. 1, all of the

in a single

peak which

["Clethacrynic

coincided

acid remaining

with the glutathione

bound to liver cyto-

z-transferases

peak as

Sephadex G-100 elution of liver cytosol from six rats killed 90 min following administration of P'Clethacrynic acid (2 mg/kg, sp. act. 2 &i/mg). Sixty ml of cytosol were chromatographed on a column (5.0 x 100 cm) using 0.01 M sodium phos-

1207

Preliminary Communications

phate (pH 7.4) as elution buffer, and 10.2 ml fractions were collected. Enzyme activity, as assayed by CDNB-GSH conjugating activity (A), &s.O (e), and radioactivity (o), was measured in every other fraction.

monitored

by GSH-CDNB

of the administered

conjugating

seen in Table 1, when aliquots quential

washes

dose of

['?Yethacrynic

appeared

to covalently

Covalent

1.

The equivalent

of a pool of fractions

of trichloroacetic

cent of the radioactivity

Table

activity.

of approximately

dose of [1"Clethacrynic acid was recovered

acid, methanol,

in the original acid remained

enzyme

bound.

bind to the giutathione

binding

of [14Clethacrynic

approximately

13.5 per-

amount of ethacrynic

acid

z-transferases.

acid to rat liverglutathione~-transferases* acid bound % Initial

pg/mg protein

fractions

1.76 + 0.02

acid

After methanol After chloroform (amount covalently

As

pool or 2.7 percent of the administered

[l!C]ethacrynic

After trichloroacetic

peak.

from this peak were treated with se-

and chloroform,

Thus, a substantial

Fraction

Pooled transferase

20 percent

in the transferase

100

0.56 f 0.02

31.8 _c 1.2

0.25 * 0.02

14.3 + 1.2

0.24 f 0.01

13.5 f. 0.3

bound)

*Six aliquots (2 ml each) of a pool of Sephadex G-100 eluates containing enzyme activity (Fig. 1) were precipitated with 0.5 ml of 50% trichloroacetic acid and then treated sequentially with trichloroacetic acid, methanol, and chloroform until no further radioactivity could be washed off. The precipitates were then dissolved in 1 ml of I N NaOH and radioactivity was measured.

With

further

purification,

AA being distinct activities reaction

(Fig. 2).

whereas

of ethacrynic

elute before

Fig. 2.

closely

conforms

negligible

activity

to the descriptions

Binding

of

acid to glutathione

the salt gradient

[14Clethacrynic

were separated,

transferases

in Fig. 2 labeled A and C also catalyzed

eluted with the first peak of GSH-CDNB

lective binding which

The fractions

transferases

C and A by virtue of their GSH-methyl-iodide

peaks B and AA showed

tion of transferases radioactivity

the various

from transferases

contained

(not shown).

conjugating

no radioactivity

acid to glutathione

activity, C.

the DCNB-GSH

Thus, the separa-

by Habig -2 et al

z-transferase

B and

conjugating

(2).

All of the

suggesting

Transferases

se-

D and E

(not shown).

z-transferase

C. Fractions

82-96

Preliminary Communications

1208

from Fig. 1 were pooled, batch adsorbed with DEAE-cellulose, and then chromatographed on a CM-cellulose column (1.5 x 40 cm) and eluted with a 100 ml linear gradient of 0.01 M potassium phosphate (pH 6.7) containing 0 to 0.2 M KCl. Each fraction was assayed for conductivity (upper figure), iodomethane-GSH conjugating activity (a), CDNB-GSH conjugating activity (A), and radioactivity (0).

DISCUSSION The glutathione potential variety

S-transferases

functional

of substrates

including

binding many non-substrate to the hypothesis

have three major characteristics

importance:

(1) they catalyze

many drugs and carcinogens

ligands

teins; and (3) they form covalent cinogens

interactions

detoxification

with the glutathione (1,3).

proteins.

scribe additional A substantial

interactions

enzymes

Since

acid-GSH

ligands

strate binding non-substrate

conjugating

activity

sites

for other

at its binding

we cannot exclude

timate that at most one-third cule of ethacrynic

acid bound per molecule

tained by our transferase Previous than

95

studies

C fractions

The apparent

C as a non-substrate

of enzyme.

suggest

speciof

may function

ligand

that ethacrynic

acid arlyated

activity.

activity

remaining

of ethacrynic

the

We es-

only 1 mole-

GSH-CDNB

for by the enzyme

as acid

in a similar

bound, assuming

Thus, the ample

that non-

from the sub-

that ethacrynic

and loss of enzyme

injection

by

AA and B, althoughtrans-

It is conceivable

C was covalently

z-transfer-

since the majority

Recent studies

could be accounted

re-

unbound.

acid, greater

from the plasma and that the bulk of etha-

in the liver is unconjugated.

acid in the liver at 90 min.

binding deL-transferases.

at sites separate

the possibility

role as

to interact

for GSH-conjuga-

for one of the transferases

(3) have shown that, 90 min after

crynic acid retained

car-

acid is activated

is unique. interest

in transferases

destruction

percent of the drug has been cleared

tered ethacrynic

binding

(2,13).

of transferase

of certain a potential

as a substrate

that ethacrynic

(2,ll).

z-transferase

site with resultant

pro-

acid and the glutathione

-S-transferases

transferases

to glutathione

Alternatively,

fashion.

activity

(8,12) and that substrates

ligands

metabolites

thus serving

(8) giving rise

acid bound to the glutathione

is present

of a wide

or storage

and, in part, covalent

C is of particular

bind to the glutathione

wascovalentlybound

enzyme

ethacrynic

this covalent

for transferase

ferase C does have some of this enzyme substrate

selective

there is no evidence

prior to binding,

ficity of this binding ethacrynic

between

green

carriers

acid has been shown previously

(13.5%) of ethacrynic

proportion

(9,10),

in the first capacity,

studiesindicating

ases was bound covalently. microsomal

Ethacrynic

&transferases

Our present

transport

with reactive

and azo-dyes

GSH-conjugation

(1,2); (2) they are capable of

and indocyanin

as intracellular

such as 3-methylcholanthrene

important

tion

such as bilirubin

that they function

that give rise to their

the enzymatic

We recovered

This suggests

20 percent

that ethacrynic

of the adminisacid binding

in the, liver in part reflects -in vivo storage of the drug. ACKNOWLEDGEMENTS--We are indebted to Mr. John Kuhlenkamp for expert technical assistance and This research was supported by VA research funds Ms. Melanie Lee for typing the manuscript. and US Public Health Service Grant GM/CA 24987. REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13.

E. Boyland and L.lF. Chasseaud, Adv. Enzymol. 32, 172 (1969). W.H. Habig, M.J. Pabst and W.B. Jakoby, J. biol . Chem. 249, 7136 (1974). rma. exa. , Ther. 205, 471 (1978). J.D. Wallin, G. Clifton and N. Kaplowitz, J. Pha..... 193, 265 (1951). O.H. Lowrv. N.J. Rosebrouah. A.L. Farr and R.J. Randall..J biolxhem. J. Booth,"E. Borland and F.-Sims, Biochem. J-79, 516 (1961). M.K. Johnson, Biochem. J. 9&, 44.(1966;). N. Kaplowitz, J. Kuhlenkamp and G. Clifton, Biochem. J. 146, 351 (1975). m._-. 250_ 8670 (1975). J.N. Ketley, W.H. Habig and W.B. Jakoby, J. biol. Chew... S. Singer and G. Litwack, Cancer Res. XL, 1364 (1971l. 122, 53~ (i971j. B. Ketterer and 0. Beale, Biochen 1.. lakoby, Archs Biochem. Biophys. 175, 710 (1976). W.H. Habig, M.J. Pabst andM.M. Bhargava, I. Listowsky and I.M. Arias, -~ J. b iol. Chem. 253, 4112i~~~~B). 2 193 (: Y. Sugiyama, T. Iga, S. Awazu and M. Hanano, J. Pharm, -9