A new potent inhibitor of lipid peroxidation in vitro and in vivo , the hepatoprotective drug anisyldithiolthione

Vol.

No. 3, 1986

135,

March

28.

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS Pages

1986

A NEWPOTENT INHIBITOR

1015-l

021

OF LIPID PEROXIDATION IN VITRO AH) IN~VI-VO, DRUG ANISYLDIT+HIOLtHIONE

THE HEPATOPROTECTIVE Daniel

MANSUYa

a Laboratoire

de

, Amor

SASSIa

Chimie

et

, Patrick

Eliochimie

Received

Fehruary

DANSETTEa

Pharmacologiques

rue des Saints Peres, Laboratoire de Chimie Therapeutique, Baptiste-Clement, 92290

75270

45

b

M.

Paris

Faculte Chatenay-Malabry,

and

PLATb

Michel

et Toxicologiques, Cedex 06, FRANCE de Pharmacie, rue JeanFRANCE

12, 19.86

The drug anisyldithiolthione (AOT) acted as a good inhibitor of lipid peroxidation induced in rat liver microsomes either chemically by FeS04 and reducing agents (cysteine or ascorbate) or enzymatically by NAOPH and CC1 . AOT was found as potent as propylgallate with IC50 around w and much more potent than vitamin E and levamisole. ADT was also found as a good inhibitor of ethane exhalation by rats treated by NO.7 mg perkg). CC1 (ID50d5mg per kg) and by mice intoxicated by acetaminophen (ID At ti oses as low as 5 mg per kg it completely suppressed ethane exhala 5P. ion by acetaminophen-intoxicated mice and also protected them very efficiently against mortality caused by acetaminophen overdose. The inhibitory effect of AOT toward lipid peroxidation seems to be linked to the presence of its dithiolthione function. D 1986 Academic Press, Lnc. It stress

dative 02

is now well characterized occurs

reduction

tive

stress

is

tion

of

cell

the

several

non-toxic

prevent

the

in

the

by many

as

compounds

tect

has

been

mice

against

Blanpin

and

results

showing

ethane also by

of

the

mg/kg

that

years

many acute

that

AOT in

low

effects

of and

inhibitor

the

toxicity

induced

seems

interesting

in vivo

degradaby

to

in

order

to

animals

intoxicated

AOT

only

either

inhibit

prevent

was recently

ref.5).

ethane

mortality

peroxidation Ccl4

exhalation caused

described

and Ccl4

This

by

drug

a

acetaminophen

of lipid

by

also

from

oxida-

thus

peroxidation

agent

results

of

but

this

peroxidation.

vivo

not

of

slow

in

oxi-

derived

species

5-(p-methoxyphenyl)-3H-l,Z-dithiol-3-thione,

doses

acetaminophen

lipid

an

more or less

a

(3,4).It

inhibit

hepatotoxic

a good

in

involved

as a choleretic

unpublished

is

are

of

consequence

resulting

processes

situations,

production

general

A

lipids

this

pathological

intense

(I).

to strongly (AOT),

Christen,

exhalation shows

500

M.O.

and

cells

several

or acetaminophen

(2)

effects

for

used

unusual of

Such Ccl4

able

deleterious

during

of cell

Anisyldithiolthione

which

an

peroxidation such

that

kinds

organization.

chemicals

find

recognized

by

paper

(0.

reports in vitro

to proFoussardfirst and

or acetaminophen. by

mice

acetaminophen

of

It intoxicated overdose.

MATERIAL AN) METHODS Chemicals: AOT (SulfarlemR) was kindly supplied by L.T.M.(Suresnes, France) ; 5-(p-methoxyphenyl)-3H-l,Zdithiol-3-one (ADO) was prepared by oxidation of ADT by mercuric acetate as described previously (6). (Il-tocopherol (vit.E) and levamisole were purchased from JANSSEN CHIMICA and ethane N30 from AIR LIQUIDE (France). Propylgallate was a gift of Or. Willson (U.K.). 0006-291X/86 1015

All

Copyrighi Q 1986 rights c~f’ wproduuion

$1.50

by Academic Press, Inc. in any ,fiwm reserved.

Vol.

135,

No. 3, 1986

Animals

BIOCHEMICAL

: Male

from IFFA CREDO per kg each day

Sprague-Dawley

(France). for three

AND

(SD)

Liver

rats

and

RESEARCH

female

NMRI

COMMUNlCATlONS

Swiss

mice

were

purchased

microsomes from rats pretreated by phenobarbital were prepared as already described (7).

days)

: a/measurement

Assays

BIOPHYSICAL

lipid

of

peroxidation

(80 mg

in vitro

a.l/ Peroxidation induced by FeSO and either cysteine or ascorbate : 1 mg of microsomal proteins measured by the Lo&v method (8) were susoended in Iml Tris HCl buffer 35 mM,’ pH : 7.4, containing KC1 0.1-M. Peroxidation was initiated by addition of 500pM cysteine or ascorbic acid and 5p ferrous sulfate. Incubation-time was 30 min for the cysteine-ferrous sulfate system and 15 min for the ascorbic acid-FeSO system. Peroxidation was stopped by adding the TBA reagent (2 ml ice cold solution 8f 15X trichloroacetic acid, 3.25 mM thiobarbituric acid and 0.25 M HCl).Samples were then incubated for 25 min in a boiling water bath, cooled, centrifuged, and the absorbance of the malondialdehyde-TBA adduct was read at 535 nm (9). Inhibitors were added to the microsomal suspension prior to the addition of ferrous sulfate. a.2/Peroxidation

induced

orotein/ml) presence

were

of cose-6-phosphate

started

by

oerformed

0.1

mM EDTA and and 2 units

by addition of 5 ~1 was measured

malondialdehyde

of

: Microsomal incubations (1 mg buffer, pH : 7.4, in thr 1 mM NADP, 10 mM gluPeroxidation was min incubation at 37°C

of Ccl4

M potassium phosphate a NADPH generating system containing of glucose-6-phosphate dehydrogenase. After 10 of Ccl4 1 M in ethanol.

0.1

as described

b/measurement of

above.

lipid

peroxidation

in vivo

(IO)

by mice treated with acetaminophen : Female NMRI Swiss were starved for 72 hours in order to deplete hepatic (3,4$, received either 0.5 ml of acetaminophen (20 mg/ml) in 019% containing 5X dextran, or only NaCl solution for controls. ADT was injected 3h before acetaminophen in 0.5 ml corn oil. Control mice either treated or not by acetaminophen were also injected with the same volume of corn oil. For ethane determination, four mice were kept in a 100 mm diameter dessicator (Jencons, GB), containing 809 of soda-lime (anesthesic grade) for CO2 adsorption. The dessicator was connected to an oxygen reservoir at atmospheric pressure through a three way stopcork and a gas valve (oil trap). Three hours after the beginning of the experiment, thr stop-cork was inverted, air within the dessicator was stirred by using a 60 ml syringe 50 ml were removed and aliquots (6ml) were injected in a Packard model 427 gas chromatoAnalysis were run at 150°C using a 2 m X 2 mm stainless graph for ethane determination. steel column filled with carbosieve G 60/80 mesh, 25 ml/min helium as carrier gas and a Calibration was performed by injecting known amounts of flame-ionisation detection. ethane in the dessicator. b.l/measurement mice (20 o glutathio;e NaCl solution

exhaled

reduction

enzymatic

in 1 ml

of

averaae

ethane which (i.p.)

weiaht)

of exhaled ethane by Ccl4 treated rats : Male (SD) rats (200 g 1 ml per kg of Ccl4 dissolved in corn oil (200 ~1 CC1 weight) received i.p. in 1 ml of corn dil + 800 ~1 corn oil) and appropriate doses of ADT were injected i.p. 3 hours before CC1 administration. Control rats intoxicated or not with CC14 received the same amoun i! s of corn oil.Each rat was kept in a 150 mm diameter dessicator containing 250 g soda-lime and exhaled ethane was measured after three hours with the method described above for the mice. b.2/measurement

average

Inhibitory dation

induced

ferrous salt concentrations were

effects

around

of in

rat

associated

(IC50) 3pM.

AD1 on lipid liver with leading

Analogous

RESULTS peroxidation

microsomes to

50%

classical

by

a reducing

in vitro

agent

chemical

systems

such as cysteine

inhibition

IC50 values,

inhibits

:ADT

in thepM

of

were

(MDA) found

peroxi-

consisting

or ascorbate

malondialdehyde

range,

lipid

for

in (11).

formation ADT

inhibi-

a The

Vol.

135,

No. 3, 1986

Table

I:

Inhibition

(malondialdehyde

BIOCHEMICAL

AND

by ADT derivatives

or

formation)

induced

BIOPHYSICAL

by

antioxidants

or

enzymatically

chemically Lipid FeS04

RESEARCH

peroxidation

of

peroxidation

rat

liver

microsomes

FeSO4

+ Cysteine

lipid in

in

COMMUNICATIONS

microsomes

induced

+ Axlorbate

by

CC14

+ NADPH

INHIBITORS ( 10%)

‘C50

(a)

HO HO

/

\

-

;-“a

4.0

:

0.1

1.3 + 0.3

3.0

_+ 0.2

0

+HO n-propylgallate

IO0

loo

loo

levamisole Vitamin

E

50

3.0

,,“&s

ADT

~~30~o

A’,0

(aI

Mean

values

of

lipid

peroxidation

Ccl4

in

the presence of

bed

as

(12)

of

protective

a

very

exhibited

Interestingly,

NADPH

(2)

ADO

(vitamin

induction

of

effects derived

almost

of ADT against lipid

Control

rats

no

only

received After

same time

fatty

acid

peroxidation

before

that

of Ccl4

tance,

when

100

markedly

2.0

z 0.1

>I00

mg per decreased

kg

a level

classical or to

the

has been descri-

and hepatotoxicity (Table of

lipid

between

or

chemical

which

its

1). C=S

function

peroxidation

IO -6M

and

by

induced

10s4M

(Table

induced in vivo in rats by CChtreatment

oil

increased

ADT was to

upon

(IC50>100~)

toward

of

(n-propylgallate)

peroxidation

effect

reduction

conditions,

Levamisole,

in vitro

i.p.

treatment

important

enzymatic

depending

at concentrations

corn

by

from ADT by replacement

exhaled

by Ccl4 a factor

by

and fragmentation an

50~M

lipid

inhibitory

peroxidation

per kg).

the

ethane

0.1

identical

peroxidation).

inhibitory

Effects

within

: around

E

lipid

poor

or enzymatically,

exhaled

+

IC5-, values

to similar

CC14-induced

compound

microsomes Under

against

the

having

1).

agent

either

chemically

liver

(table

values

showed

nmoles

3.0

>I00

rat

lead

a C = 0 function,

(12.521.5

in

peroxidation

higher nature

_+ 0.1

“*

induced of

lipid

considerably enzymatic

loo

z SE ( 4 experiments)

tion

inhibitors

_+ 25

(13)

decrease

of

administered identical

1017

i.p. to that

amounts

of

ml per kg i.p.)

of

(Fig.1). ethane

low

(I

as a result

3

within

exhaled

of

was observed. 3 and

observed

for

6 h

before

control

:

unsaturated

administration

Upon

formation between

ethane

ethane

1).

of

ADT

For insCC14, rats

(Fig.1).

3 h

Vol.

135,

No. 3, 1986

BIOCHEMICAL

AND

BIOPHYSICAL

I

I

RESEARCH

I

/

5 12.5 25 50 DOSES Figure

1

:

Protective

effects

exhalation)

in

Amounts of

values

order

ADT of

to

and

determine

ethane

ADT

at

exhaled

per kg).

(1

ml

is

already

As

kg

and

then

(3

h)

in

peroxidation

CC14

(3

(measured

: a dose-response

exhaled

during

after)

h

by

by

ethane

relationship.

rats

3 h by

relationship

dose-response

treated

by

before

Ccl4 the

Fig.1,

around

(m&g) lipid

by

I 101

CC14

(1

treated

ml

various

by

per

kg).

Mean

animals).

rats

shown

ADT

against

ethane

(i.p.) (3

by

time

complete

per

10 mg

SE

the

a fixed

of

ADT

2

ADT

rats treated

(nmoles)

doses

In

of

OF

COMMUNICATIONS

20 mg per

between

rats

CC14, injection

and

inhibitory

kg,

were

amounts

pretreated

of

by

a given

dose

of

ADT

starts

at 5

half-maximum

effect

administered

by various

then

effect

the

the

being

doses

of

Ccl4

per kg and

mg

obtained

with

ADT.

Effects of ADT on lipid peroxidation and toxicity induced in vivo in mice by acetaminophen : As already described (3,4) untreated NMRI Swiss mice but also mice treated by up

to

these

500

mg per

kg

acetaminophen

mice

had

been

starved

became

could

of

mice

by

ADT

doses

not

treated

Whereas led of

kg

easily

ADT as

3

low by

i.p.

effect acetaminophen

to

by

5 mg

acetaminophen

of

inhibition

propylgallate

(table

2).

against

This

between

by

per

As

a

of

ADT

was to

of ethane

exhalation,

administration

led

to a partial

inhibition

of

IO

ethane

hepatotoxicity. all 24 h,

and

1018

exhalation

obtained

was

Whereas all mice pretreated

of

If

mice

2,

pretreatment

exhalation. to

With

that

with

of

accompanied

mice treated by 50pmoles

mice

0.7 mg per

kg

acetaminophen

of identical inhibition

ethanc

either

in

mice 3 h before

or levamisole

only

Fig.

in

3h. they

amounts

ethane

reduced ethane level identical

effect kg

large of

shown

within

ethane glutathione,

exhaled

levels

ADT).

administration decreased to

of

hepatic

and

half-maximum

20pmoles

acetaminophen died

not

amounts

deplete

control

see

but

the

to

low

toxicity

(Fig.2,

ethane

kg, and

administration

E,

(3)

acetaminophen

per

very

72 h in order

acetaminophen

h before

only

acetaminophen

detected

as

to a complete vitamin

formation tive

be

or treated

untreated

for

more sensitive

much

which

exhaled

amounts of

by

ethane a

protec-

by 500 mg per (12 mg) per

Vol.

135,

BIOCHEMICAL

No. 3, 1986

z

600

400-

$

,oq-

=”

zoo-

o

E

Protective

COMMUNICATIONS

lo+

z a E

’

,/‘I’ ,

effects

ethane

RESEARCH

]

yM

0”

2 :

BIOPHYSICAL

500 -

$

Figure

AND

exhalation)

-’

0.1

0.5

I

DOSES

OF

ADT

of

ADT

in

mice treated

against

5

in

12.5

100

(mglkg) vivo

lipid

peroxidation

(measured

by

: a dose-response

acetaminophen

by

relationship. Amounts

kg kg,

ADT

before

ADT

(nmoles)

then

treated

i.p.

acetaminophen.

of

by

far

(70% survival

were

of

25,

alive

0 and

within

of

ADT

+

values

3 h by

(i.p.)

24 h.

agent

starved

mice

and

3 h after

during

by

500

mg

72 per

h, kg

(8 animals).

SE

after

protecting

a better

instead

exhaled doses

Mean

acetaminophen

is

ethane

various

by

As

shown

in

than

vitamin

E,

0% respectively

for

these

table

2,at

20 pmoles

propylgallate

per

or levamisole

compounds).

DISCUSSION

results

The

protect

ADT to

Table

of

table

against

animals

2 : Inhibitory

the

effects

effects of ADT and several hepatotoxicity induced by

antioxidants acetaminophen

Inhibition

(20 pmoles

per

ethane

kg)

None

(b) (4

hfilce

100

( 50 poles Vitamin E

per

of

kg)

the

Ccl4

or

on in vivo (a) in mice

of

remarkable

on

lipid

% survival

after

0 96

%

%

(IO)

100%

100

%

(5)

W%

25

%

(4)

%

0

% (5)

%

0

96

600

z

nmotes

45

of mice

72

of used

h received

pmoles

per

(Lp.)

ethane in

20 per

the

kg

per

(c)

h

(8)

70

40

for

and

exhalation

40

acetaminophen

24

of They

peroxidation

Levamisole

starved mg/kg

ability

acetaminophen.

Propylgallate

500 number

(5)

0 % fb)

ADT

(a)

data

hepatotoxic

COMPOUNDS

ADT

previous

2 confirm

3 h

after

experiment.

1019

kg

of

the

acetaminophen

compound

(1.p.) administratlon.

(5)

and

then

(3

h

after)

Vol.

135,

No. 3, 1986

show

that

Upon

administration

mg per

ADT

kg)

kg(5)),

exerts

its of

(data

not

3 h before

protection

protective

shown) doses

also

animals

treated

show

that

peroxidation

minophen

by

(IDs0~0.7

per

mg

these

compounds,

mice against

the

hepatotoxic

far

as the

As

has been

previously (5).

tor

peroxidation

of

lipid

inhibition to

an

The

equally

to

tion

and of

lipid

peroxidation

lipid

peroxidation,

portant

role

in

be

involved

lipid

important man since

this effects

ACKNOWLEDGWNTS and

financial

also in

aceta-

or

antioxidants (Table

2).

When

to protect

ADT

on

content

ADT

is

after

peroxidation

The

fact

that

by

Fe(II)

and

of

lipid

partly

involved Ccl4

and

as a better similar

its

in

ADT was

found

either

cys-

observed

inhi-

In this

NADPH.

(and)

by

peroxida-

inhibitor

than

those

obtained

to

This

(2)

inhibitor

an

Ccl4 inhibi-

or

*Ccl3

into

it

a strong

lipid

Ccl4

chemically

range

GSH

concerned,

microsomes.

really by

is

ADT

liver

reductase.

found

thepM

are its

drug

“antioxidant”

against

still

of

to appear obtained

has been

levels

required

because

in

used for

its

(ADO is

vivo

at

of

very

at least

vitro

in

vitamin

E

with

play of

the

other

ADT

dithiolthione

is

effects an

low doses

and

without

of

which

(Table (iii)

Ccl4

im-

and

ADT could

interesting

function

not an inhibitor)

an

However,

GSH consumption.

Anyway,

40 years

could

ADT

manifestations and

to know whether role.

(i)

effect

hepatotoxic

hydroperoxide

hepatoprotective

the inhibition also

the

effect lipid

inhibitor

effects

least

by

that

rat

in

of

microsomes

in

that

protective

to explain for

inhibitory

values

suggest

reducing

peroxidation

adverse

induced

experiments

supplementary

at

is

by

vivo

propylgallate.

its by

is

ADT was

results

acetaminophen,

ADT

in

of

show

of

induced

that

effect

gave ICso

antioxidant

effect

P-450

of

of

decrease

NADPH

cytochrome

exhalation

ability

effect the

activation

Our

overdoses.

clearly and

mg per a complete

ethane

or in mice

(24

h,

inhibitor described

kg.

(500 later.

week

more pronounced

prevents Ccl4

on

per

at doses 24

or levamisole

even

the

shows

this

for

These

by

within

one

previously

propylgallate

results

or orally

a good

hepatoprotective

ADT

ZOfimolea

previously

per kg)

acetaminophen

peroxidation 1)

bition

well-known

and

lipid

(Table

It

this

as

die

even

is

WlOmg

an

low

used mice

effect

than

E,

toward

ADT

P-450

that

levamisole.

better

of

that

of

assay and

is

as

COMMUNICATIONS

2)

alive

ADT

CC14(IDs0

effects

of

all

inhibitory

exhibits

induced

stop

suggests

those

which

to an “antioxidant”

cytochrome potent

than

for

by

vitamin

ADT

origin

effect

or ascorbate

teine

lower

mice being

aforementionned

may be due inhibitory

microsomal

as

reported

intoxication

It

kg).

doses

kg)(Table

mg

a strong

rats

such

to

at

or acetaminophen.

in

agents

compared

exerts Ccl4

RESEARCH

per

(12

pretreated

induced

hepatoprotective

i.p.

acetaminophen

ADT

8lOPHYSlCAL

already

considerably of

all

either

AND

effect

either

ADT,

was observed,

results lipid

BIOCHEMICAL

its

significant

I),

new seems

(ii)

possible

toxic

its use

or

.

: We thank

support.

Laboratories

We also

thank

L.T.M.

Dr.

Marcel 1020

(Suresnes,France) DELAFORGF for

for his

their

scientific

scientific assistance.

in

Vol.

135,

No. 3, 1986

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

REFERENCES 1

Sies,

2

Recknagel, in

H.

Oxidative

stress

R.O.,Glende,

carbon

vol. 3

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III,

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Jr.

E.D.,

tetrachloride

and

toxicity.

97-132,

Academic

l-8

p.

311-330,

Hruszkewycz,

“Free

In Press,

New

A.,

Feuerstein,

S.,

and

Wendel,

A.,

Feuerstein,

S.,

(1981).Biochem.

Ansher,

S.S.,

Wendel,

and

Konz,

Academic A.M.

radicals

Press,

(1977).

London.

Chemical

in biology”.

Mechanism

Pryor,

(W.A.

ed.),

York.

K.H.

(1979).

Pharmacol.

Biochem.

-13 p.

2051-205

Bottcher, Kremers, and 8

10

P.,

O.H.,

P.,

and

(1948).

Chem.

Beaune,

Ph.,

J.

Gielen,

Lowry,

193, 9

Dolon,

B.

(1981).

Bueding

Ber.

Pharmacol.

E.

-81

p.

Cresteil,

(1983).

de

J. Biochem.

Eur.

N.J.,

Farr,

Sinnhuber,

R.O.

(196415.

Hepatology

p. 2531-2520 2

p.

932-935

376 T.,

Rosebrough,

E,

118

A.L.,

Graeve, p.

J.,

S.,

Collumelli,

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