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Vol. 135, No. 3, 1986 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS March 28. 1986 Pages 1015-l 021 A NEWPOTENT INHIBITOR OF LIPID PEROXIDATION IN VITRO AH) IN~VI-VO, THE HEPATOPROTECTIVE DRUG ANISYLDIT+HIOLtHIONE Daniel MANSUYa , Amor SASSIa , Patrick M. DANSETTEa and Michel PLATb a Laboratoire de Chimie et Eliochimie Pharmacologiques et Toxicologiques, b 45 rue des Saints Peres, 75270 Paris Cedex 06, FRANCE Laboratoire de Chimie Therapeutique, Faculte de Pharmacie, rue Jean- Baptiste-Clement, 92290 Chatenay-Malabry, FRANCE Received Fehruary 12, 19.86 The drug anisyldithiolthione (AOT) acted as a good inhibitor of lipid peroxi- dation 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 levami- sole. ADT was also found as a good inhibitor of ethane exhalation by rats treated by At ti CC1 (ID50d5mg per kg) and by mice intoxicated by acetaminophen (ID NO.7 mg perkg). oses as low as 5 mg per kg it completely suppressed ethane exhala ion by acetamino- 5P. phen-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 is now well recognized that during several pathological situations, an oxi- dative stress characterized by an unusual and intense production of species derived from 02 reduction occurs in many kinds of cells (I). A general consequence of this oxida- tive stress is the peroxidation of cell lipids resulting in a more or less slow degrada- tion of the cell organization. Such processes are involved in the toxicity induced by several chemicals such as Ccl4 (2) or acetaminophen (3,4).It thus seems interesting to find non-toxic compounds able to strongly inhibit lipid peroxidation in vivo in order to prevent the deleterious effects of this peroxidation. Anisyldithiolthione (AOT), 5-(p-methoxyphenyl)-3H-l,Z-dithiol-3-thione, a drug which has been used for many years as a choleretic agent was recently described to pro- tect mice against the acute hepatotoxic effects of acetaminophen and Ccl4 (0. Foussard- Blanpin and M.O. Christen, unpublished results and ref.5). This paper reports first results showing that AOT is a good inhibitor of lipid peroxidation in vitro and of ethane exhalation in vivo by animals intoxicated either by Ccl4 or acetaminophen. It also shows that low doses of AOT not only inhibit ethane exhalation by mice intoxicated by 500 mg/kg acetaminophen but also prevent mortality caused by acetaminophen 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 mer- curic 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 $1.50 1015 Copyrighi Q 1986 by Academic Press, Inc. All rights c~f’ wproduuion in any ,fiwm reserved.

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

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Page 1: A new potent inhibitor of lipid peroxidation in vitro and in vivo, the hepatoprotective drug anisyldithiolthione

Vol. 135, No. 3, 1986 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

March 28. 1986 Pages 1015-l 021

A NEW POTENT INHIBITOR OF LIPID PEROXIDATION IN VITRO AH) IN~VI-VO, THE HEPATOPROTECTIVE DRUG ANISYLDIT+HIOLtHIONE

Daniel MANSUYa , Amor SASSIa , Patrick M. DANSETTEa and Michel PLATb

a Laboratoire de Chimie et Eliochimie Pharmacologiques et Toxicologiques,

b 45 rue des Saints Peres, 75270 Paris Cedex 06, FRANCE

Laboratoire de Chimie Therapeutique, Faculte de Pharmacie, rue Jean- Baptiste-Clement, 92290 Chatenay-Malabry, FRANCE

Received Fehruary 12, 19.86

The drug anisyldithiolthione (AOT) acted as a good inhibitor of lipid peroxi- dation 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 levami-

sole. ADT was also found as a good inhibitor of ethane exhalation by rats treated by

At ti CC1 (ID50d5mg per kg) and by mice intoxicated by acetaminophen (ID NO.7 mg perkg).

oses as low as 5 mg per kg it completely suppressed ethane exhala ion by acetamino- 5P.

phen-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 is now well recognized that during several pathological situations, an oxi-

dative stress characterized by an unusual and intense production of species derived from

02 reduction occurs in many kinds of cells (I). A general consequence of this oxida-

tive stress is the peroxidation of cell lipids resulting in a more or less slow degrada-

tion of the cell organization. Such processes are involved in the toxicity induced by

several chemicals such as Ccl4 (2) or acetaminophen (3,4).It thus seems interesting to

find non-toxic compounds able to strongly inhibit lipid peroxidation in vivo in order to

prevent the deleterious effects of this peroxidation.

Anisyldithiolthione (AOT), 5-(p-methoxyphenyl)-3H-l,Z-dithiol-3-thione, a drug

which has been used for many years as a choleretic agent was recently described to pro-

tect mice against the acute hepatotoxic effects of acetaminophen and Ccl4 (0. Foussard-

Blanpin and M.O. Christen, unpublished results and ref.5). This paper reports first

results showing that AOT is a good inhibitor of lipid peroxidation in vitro and of

ethane exhalation in vivo by animals intoxicated either by Ccl4 or acetaminophen. It

also shows that low doses of AOT not only inhibit ethane exhalation by mice intoxicated

by 500 mg/kg acetaminophen but also prevent mortality caused by acetaminophen 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 mer- curic 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 $1.50

1015 Copyrighi Q 1986 by Academic Press, Inc.

All rights c~f’ wproduuion in any ,fiwm reserved.

Page 2: A new potent inhibitor of lipid peroxidation in vitro and in vivo, the hepatoprotective drug anisyldithiolthione

Vol. 135, No. 3, 1986 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNlCATlONS

Animals : Male Sprague-Dawley (SD) rats and female NMRI Swiss mice were purchased from IFFA CREDO (France). Liver microsomes from rats pretreated by phenobarbital (80 mg per kg each day for three days) were prepared as already described (7).

Assays : a/measurement of lipid peroxidation in vitro

a.l/ Peroxidation induced by FeSO and either cysteine or ascorbate : 1 mg of micro- somal 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. Peroxi- dation was stopped by adding the TBA reagent (2 ml ice cold solution 8f 15X trichloro- acetic 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 malon-

dialdehyde-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 by enzymatic reduction of Ccl4 : Microsomal incubations (1 mg

orotein/ml) were oerformed in 1 ml of 0.1 M potassium phosphate buffer, pH : 7.4, in thr presence of 0.1 mM EDTA and a NADPH generating system containing 1 mM NADP, 10 mM glu- cose-6-phosphate and 2 units of glucose-6-phosphate dehydrogenase. Peroxidation was started by addition of 5 ~1 of Ccl4 1 M in ethanol. After 10 min incubation at 37°C malondialdehyde was measured as described above.

b/measurement of lipid peroxidation in vivo (IO)

b.l/measurement of exhaled ethane by mice treated with acetaminophen : Female NMRI Swiss mice (20 o averaae weiaht) which were starved for 72 hours in order to deplete hepatic glutathio;e (3,4$, received (i.p.) either 0.5 ml of acetaminophen (20 mg/ml) in 019% NaCl solution 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 stop- cork 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 chromato- graph for ethane determination. Analysis were run at 150°C using a 2 m X 2 mm stainless

steel column filled with carbosieve G 60/80 mesh, 25 ml/min helium as carrier gas and a flame-ionisation detection. Calibration was performed by injecting known amounts of

ethane in the dessicator.

b.2/measurement of exhaled ethane by Ccl4 treated rats : Male (SD) rats (200 g average weight) received i.p. 1 ml per kg of Ccl4 dissolved in corn oil (200 ~1 CC1 + 800 ~1 corn oil) and appropriate doses of ADT were injected i.p. in 1 ml of corn dil 3 hours before CC1 ved the same amoun i!

administration. Control rats intoxicated or not with CC14 recei- 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.

RESULTS

Inhibitory effects of AD1 on lipid peroxidation in vitro :ADT inhibits lipid peroxi-

dation induced in rat liver microsomes by classical chemical systems consisting in a

ferrous salt associated with a reducing agent such as cysteine or ascorbate (11). The

concentrations (IC50) leading to 50% inhibition of malondialdehyde (MDA) formation

were around 3pM. Analogous IC50 values, in thepM range, were found for ADT inhibi-

Page 3: A new potent inhibitor of lipid peroxidation in vitro and in vivo, the hepatoprotective drug anisyldithiolthione

Vol. 135, No. 3, 1986 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Table I: Inhibition by ADT derivatives or by antioxidants of lipid peroxidation

(malondialdehyde formation) induced chemically or enzymatically in rat liver microsomes

Lipid peroxidation in microsomes induced by

INHIBITORS

HO

HO

+-

/ \ -

;-“a

0 HO

n-propylgallate

FeS04 + Cysteine FeSO4 + Axlorbate CC14 + NADPH

‘C50 ( 10%) (a)

4.0 : 0.1 1.3 + 0.3 3.0 _+ 0.2

levamisole

Vitamin E

IO0

50 _+ 25

,,“&s ADT 3.0 _+ 0.1 3.0 + 0.1 2.0 z 0.1

loo loo

loo

~~30~o A’,0 “* (aI Mean values z SE ( 4 experiments)

>I00 >I00

tion of lipid peroxidation induced in rat liver microsomes by enzymatic reduction of

Ccl4 in the presence of NADPH (2) (table 1). Under identical conditions, classical

inhibitors of lipid peroxidation lead to similar IC5-, values (n-propylgallate) or to

considerably higher values (vitamin E : around 50~M depending upon the chemical or

enzymatic nature of induction of lipid peroxidation). Levamisole, which has been descri-

bed as a protective agent against CC14-induced lipid peroxidation and hepatotoxicity

(12) exhibited very poor inhibitory effects in vitro (IC50>100~) (Table 1).

Interestingly, ADO the compound derived from ADT by replacement of its C=S function by

a C = 0 function, showed almost no inhibitory effect toward lipid peroxidation induced

either chemically or enzymatically, at concentrations between IO -6M and 10s4M (Table 1).

Effects of ADT against lipid peroxidation induced in vivo in rats by CChtreatment :

Control rats having only received corn oil i.p. exhaled low amounts of ethane within 3 h

(12.521.5 nmoles per kg). After treatment by Ccl4 (I ml per kg i.p.) ethane exhaled

within the same time markedly increased by a factor of 3 as a result of unsaturated

fatty acid peroxidation and fragmentation (13) (Fig.1). Upon administration of ADT

before that of Ccl4 an important decrease of ethane formation was observed. For ins-

tance, when 100 mg per kg ADT was administered i.p. between 3 and 6 h before CC14,

exhaled ethane decreased to a level identical to that observed for control rats (Fig.1).

1017

Page 4: A new potent inhibitor of lipid peroxidation in vitro and in vivo, the hepatoprotective drug anisyldithiolthione

Vol. 135, No. 3, 1986 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

I I I / I 5 12.5 25 50 101

DOSES OF ADT (m&g)

Figure 1 : Protective effects of ADT against lipid peroxidation (measured by ethane

exhalation) in rats treated by CC14 : a dose-response relationship.

Amounts (nmoles) of ethane exhaled during 3 h by rats treated by various

doses of ADT (i.p.) and then (3 h after) by CC14 (1 ml per kg). Mean

values 2 SE (3 animals).

In order to determine the dose-response relationship between the amounts of administered

ADT and ethane exhaled by rats treated by CC14, rats were pretreated by various doses

of ADT at a fixed time (3 h) before Ccl4 injection and then by a given dose of Ccl4

(1 ml per kg). As shown in Fig.1, the inhibitory effect of ADT starts at 5 mg per kg and

is already complete around 20 mg per kg, the half-maximum effect being obtained with

10 mg per kg ADT.

Effects of ADT on lipid peroxidation and toxicity induced in vivo in mice by acetami-

: nophen As already described (3,4) untreated NMRI Swiss mice but also mice treated by

up to 500 mg per kg acetaminophen exhaled only very low amounts of ethane within 3h. If

these mice had been starved for 72 h in order to deplete hepatic glutathione, they

became much more sensitive to acetaminophen toxicity and exhaled large amounts of ethanc

which could be easily detected (3) (Fig.2, see control levels of ethane in mice either

untreated or treated by acetaminophen but not by ADT). As shown in Fig. 2, pretreatment

of mice by ADT 3 h before acetaminophen administration reduced ethane exhalation. With

ADT doses as low as 5 mg per kg, ethane decreased to a level identical to that of mice

not treated by acetaminophen and the half-maximum effect was obtained with 0.7 mg per kg

Whereas i.p. administration of 20pmoles per kg of ADT to mice 3 h before acetaminophen

led to a complete inhibition of ethane exhalation, administration of identical amounts

of vitamin E, propylgallate or levamisole led only to a partial inhibition of ethane

formation (table 2). This inhibition of ethane exhalation was accompanied by a protec-

tive effect against acetaminophen hepatotoxicity. Whereas all mice treated by 500 mg per

kg acetaminophen died between IO and 24 h, all mice pretreated by 50pmoles (12 mg) per

1018

Page 5: A new potent inhibitor of lipid peroxidation in vitro and in vivo, the hepatoprotective drug anisyldithiolthione

Vol. 135, No. 3, 1986 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

z 600 ]

$ 500 -

yM 400-

$ ,oq-

=” zoo-

0”

z lo+

a E o ’ /‘I’ -’ E

, , 0.1 0.5 I 5 12.5 100

DOSES OF ADT (mglkg)

Figure 2 : Protective effects of ADT against in vivo lipid peroxidation (measured by

ethane exhalation) in mice treated by acetaminophen : a dose-response

relationship.

Amounts (nmoles) of ethane exhaled within 3 h by mice starved during 72 h,

then treated by various doses of ADT (i.p.) and 3 h after by 500 mg per kg

i.p. acetaminophen. Mean values + SE (8 animals).

kg ADT before acetaminophen were alive after 24 h. As shown in table 2,at 20 pmoles per

kg, ADT is by far a better protecting agent than vitamin E, propylgallate or levamisole

(70% survival instead of 25, 0 and 0% respectively for these compounds).

DISCUSSION

The results of table 2 confirm previous data (5) on the remarkable ability of

ADT to protect animals against the hepatotoxic effects of Ccl4 or acetaminophen. They

Table 2 : Inhibitory effects of ADT and several antioxidants on in vivo lipid peroxidation and hepatotoxicity induced by acetaminophen (a) in mice

COMPOUNDS

(20 pmoles per kg)

Inhibition of

ethane exhalation % survival after 24 h

(c)

None 0 % fb) 0 96 (8)

ADT 100 % 70 % (IO)

ADT ( 50 poles per kg) 100% Vitamin E

100 % (5) W% 25 % (4)

Propylgallate 40 % 0 % (5)

Levamisole 40 % 0 96 (5)

(a) hfilce starved for 72 h received 20 pmoles per kg of the compound (1.p.) and then (3 h after)

(b) 500 mg/kg acetaminophen (Lp.)

(4 600 z 45 nmotes of ethane per kg per 3 h after acetaminophen administratlon.

number of mice used in the experiment.

1019

Page 6: A new potent inhibitor of lipid peroxidation in vitro and in vivo, the hepatoprotective drug anisyldithiolthione

Vol. 135, No. 3, 1986 BIOCHEMICAL AND 8lOPHYSlCAL RESEARCH COMMUNICATIONS

show that ADT exerts its protective effect already at doses as low as ZOfimolea per kg.

Upon administration of ADT, either i.p. (12 mg per kg)(Table 2) or orally at doses (24

mg per kg) (data not shown) considerably lower than those used previously (500 mg per

kg(5)), 3 h before doses of acetaminophen for which all mice die within 24 h, a complete

protection was observed, all pretreated mice being alive even one week later. Our

results also show that ADT exerts a strong inhibitory effect on ethane exhalation by

animals treated either by Ccl4 or acetaminophen. ADT is a good inhibitor of in vivo

lipid peroxidation induced in rats by CC14(IDs0 WlOmg per kg) or in mice by aceta-

minophen (IDs0~0.7 mg per kg). It is better than previously described antioxidants or

hepatoprotective agents such as vitamin E, propylgallate or levamisole (Table 2). When

compared to these compounds, ADT exhibits an even more pronounced ability to protect

mice against the hepatotoxic effects of acetaminophen overdoses.

As far as the origin of this hepatoprotective effect of ADT is concerned, it

has been previously reported that ADT prevents the decrease of GSH content after Ccl4

intoxication (5). The aforementionned results clearly show that ADT is a strong inhibi-

tor of lipid peroxidation induced by Ccl4 and NADPH in rat liver microsomes. This

inhibition may be due to an “antioxidant” effect of ADT on lipid peroxidation or (and)

to an inhibitory effect of ADT toward the activation of Ccl4 into *Ccl3 (2) by

microsomal cytochrome P-450 and cytochrome P-450 reductase. The fact that ADT was found

equally potent to stop lipid peroxidation induced chemically by Fe(II) and either cys-

teine or ascorbate (Table 1) shows that ADT is really an inhibitor of lipid peroxida-

tion and suggests that this effect is at least partly involved in its observed inhi-

bition of lipid peroxidation induced in microsomes by Ccl4 and NADPH. In this in vitro

assay for lipid peroxidation, ADT was also found as a better inhibitor than vitamin E

and levamisole. It gave ICso values in thepM range similar to those obtained with the

well-known antioxidant propylgallate.

These results suggest that the “antioxidant” effect of ADT could play an im-

portant role in its protective effect against hepatotoxic manifestations of Ccl4 and

acetaminophen, by reducing lipid hydroperoxide levels and GSH consumption. However,

supplementary experiments are still required to know whether other effects of ADT could

be involved to explain its hepatoprotective role. Anyway, ADT is an interesting new

lipid peroxidation inhibitor because of (i) its dithiolthione function which seems

important for the inhibition to appear (ADO is not an inhibitor) (Table I), (ii) its

inhibitory effects also obtained in vivo at very low doses and (iii) its possible use in

man since this drug has been used for at least 40 years without significant toxic or

adverse effects .

ACKNOWLEDGWNTS : We thank Laboratories L.T.M. (Suresnes,France) for their scientific

and financial support. We also thank Dr. Marcel DELAFORGF for his scientific assistance.

1020

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Vol. 135, No. 3, 1986 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

1

2

3

8

9

10

11

12

13

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1021