Studies on the systems involved in succinonitrile metabolism in liver

Pharmacological Research Communications, VoL 12, No. 5, 1980

STUDIES

ON THE

SYSTEMS

INVOLVED

433

IN S U C C I N O N I T R I L E

METABOLISM

IN L I V E R M.

Floreani,

F.

Carpenedo,

R.

Santi

and

A.R.

Contessa

I n s t i t u t e of P h a r m a c o l o g y , S c h o o l of M e d i c i n e L a r g o E. M e n e g h e t t i 2 - I 3 5 1 0 0 Padua, I t a l y

SUMMARY. The liberation of cyanide from succinonitrile has been studied "in vitro" to obtain information on the subcellular systems involved in the metabolism of the drug. I~ The complex between succinonitrile and cyt. P450 in mlcr¢somes, and the inhibition of cyanide liberation by SKF-525A in a dose-dependent way in slicos "suggest that the first step of succinonitrile metabolism takes place in endoplasmic reticulum. 2. The decreased liberation of cyanide due to oxidative pho~phorylation inhibitors sugges%s that an energy-dependent mltochondrial step plays a role in subsequent step. Therefore our data suggest that succinonitrile metabolism is a multistep process in which microsomal and mitochondrlal fractions are involved.

INTRODUCTION.

Succinonitrile

some m e t a b o l i t e s important 1973).

for

the

urine.

al.

R.

1973;

S.M.

70-90

in the

and

to t h i o c y a n a t e % of

R.

the as

and

by

cyanide

(Contessa

is c o m p l e t e l y

urine

Cavanna

1975)

toxicity

cyanide

rhodanese

About

is e l i m i n a t e d Santi

the drug

Normally,

mitochondrial

(Curry

is t r a n s f o r m e d

A.R.

is

liver

which

thiocyanate

and

Santi

R.

by

is e x c r e t e d

in

succinonitrile

(Contessa

F.

in

the m o s t

transformed

administered

Pocchlari

the

1972;

A.R.

Lodi

and

F. et

1973).

0031-6989/80/050433.-09/~02.00J0

@ 1980 The Italian Pharn'mcologlcalSoclety

Pharmacological Research Communications, Vol. 12, No. 5, 1980

434

Contemporary administration alter the liberation of c y a n i d e b l o c k i n g cyanide l i b e r a t i o n (Contessa A.R.

et al.

to a n i m a l s of o t h e r drugs can from s u c c i n o n i t r i l e .

CC14 by

s t r o n g l y lowers the d r u g t o x i c i t y

1978).

Chronic ethanol

t r e a t m e n t by

e n h a n c i n g the s u c c i n o n i t r i l e m e t a b o l i s m halves its LD50 tessa A.R. Cyanide ~'in vitro"

et al.

1978; C o n t e s s a A.R. et al.

liberation

from s u c c i n o n i t r i l e

(Con-

1978a).

can be e v i d e n t i a t e d

by only using liv e r slices b e c a u s e the f r a g m e n t a t i o n

of c e l l u l a r o r g a n i z a t i o n c o m p l e t e l y b l o c k s the c y a n i d e The purpose of this work on the s u b c e l l u l a r

systems

formation.

is to o b t a i n further i n f o r m a t i o n involved

in s u c c i n o n i t r i l e

metabolism.

M A T E R I A L S AND METHODS. Male albino W i s t a r rats (body wt 250-300 g) m a i n t a i n e d on l a b o r a t o r y diet w e r e used. The e x p e r i m e n t s were p e r f o r m e d using liver slices of normal and drug p r e t r e a t e d animals p r e p a r e d as p r e v i o u s l y d e s c r i b e d (Contessa A.R. and Santi R. 1973) or rat liver h o m o g e n a t e p r e p a r e d in 3 v o l u m e s (w/v)of 0.25 M sucrose - 0.1 Tris HCI pH 7.4 or m i c r o s o m a l fraction prepared in 0.15 M KCI - 0.02 M Tris HCI pH 7.4. Standard incubation medium. L i v e r slices (500 mg wet wt) were incubated in 4 ml K r e b s - R i n g e r p h o s p h a t e b u f f e r pH 7.4 c o n t a i n i n g 1.25 ~4 s u c c i n o n i t r i l e + 1.25 m M sodium t h i o s u l p h a t e for 2 hrs in a Dubnoff shaker at 37°C. 0.5 ml h o m o g e n a t e were i n c u b a t e d for I hr at 37°C in a total v o l u m e of 2 ml of I00 m M Tris HCI pH 7.4, 2.5 m M M g C I 2, 8 mM KCI, 0.5 m M NADP+,.-3 mM g l u c o s e - 6 - p h o s p h a t e , 2.6 I.U. G-6-P d e h y d r o g e n a s e (sigma, Type VII), 2.5 mM s u c c i n o n i t r i l e + + 2.5 ram sodium thiosulphate. I mg m i c r o s o m a l or m i t o c h o n d r i a l p r o t e i n was i n c u b a t e d for 10 min at 37°C in a final v o l u m e of I ml of the same i n c u b a t i o n m e d i u m used for h o m o g e n a t e with 5 mM s u c c i n o n i t r i l e + 5 m M sodium thiosulphate, and I mg r h o d a n e s e (Sigma). ~ h e n m i t o c h o n d r i a l f r a c t i o n was used r h o d a n e s e was omitted. A s _ ~ . C y a n i d e f o r m a t i o n was e s t i m a t e d as t h i o c y a n a t e (Contessa A.R. and Santi R. 1973). D - l a c t a t e - d e h y d r o g e n a s e a c t i v i t y w a s m e a s u r e d by the m e t h o d of J o h n s o n (Johnson M.K. 1960) in the s u p e r n a t a n t of c e n t r i f u g e d slices. The m o d i f i c a t i o n of the cyt. P450 a b s o r b a n c e s p e c t r u m induced by the drug was e s t i m a t e d f o l l o w i n g the record of a b s o r b a n c e of 2 mg m i c r o s o m a l protein sus p e n d e d in 100 m M p o t a s s i u m p h o s p h a t e

Pharmacological Research Communications, VoL 12, No. 5, 1980

435

buffer pH 7.5 from 480 to 380 nm in a spectrophotometer DB, in the absence and in the presence of the drug. RESULTS AND DISCUSSION. of succinonitrile

It has been reported

can be evidentiated

not in liver homogenate results confirmed liver microsomal fraction

subcellular

by isolated

liver mitochondrial

thiosulphate.

The complete in separate

was taken as an indication

systems are strictly

of succinonitrile

evidence

reticulum

the cyt.

of sodium

These

that

in which

involved.

with microsomal

the

The

and m i t o c h o n d r i a l

were then investigated.

A direot plasmic

and Santi R. 1973).

of the drug is a multistep process

intracellular

fractions

(Contessa A.R.

of the m e t a b o l i s m of succinonitrile

compartments

interactions

in rat liver slices but

fraction or by isolated

biotransformation several

the m e t a b o l i s m

that the drug is not transformed

in the presence

disappearance

that

Beckman

that succinonitrile

system was obtained

P450 absorbance

interacts

with endo-

by the m o d i f i c a t i o n

spectrum determined

of

by the drug in a

Type I way. Further

indication of the involvment

of endoplasmic

system in the metabolism of succinonitrile of its b i o t r a n s f o r m a t i o n

"in vitro"

action of SKF-525A at mlcrosomal (Gillette J.R.

and Sasame H.A.

reticulum

was the inhibition

by SKF-525A.

The specific

level is widely documented

1964;

R o g e r s P. and Fours J.R.

1964). Fig. added

I shows that increasing

"in vitro"

decreased

from succlnonltrile.

liver slices prepared

35 mg/Kg

i.p.

ability to transform medium

of this substance

to the incubation m e d i u m of liver slices

the formation of cyanide

Accordingly, SKF-525A

concentrations

from rats pretreated

45 min before the sacrifice

succinonitrile

with

lost the

when added to the incubation

(Table I).

From these results of succinonitrile

with

it can be concluded the endoplasmic

that the interaction

reticulum

is an important,

Pharmacological Research Communications, Vol. 12, No. 5, 1980

436

U_ 0

.

,

,=

|

. . . .

05

0

i

SKF-525A (raM) Fig.

I. The effect of increasing concentrations of SKF-525A on cyanide formation from succinonitrile in rat liver slices. Liver slices (5.00 mg wet wt) were incubated in 4 ml Krebs-Ringer phosphate buffer pH 7.4 containing 1.25 m M succinonitrile + 1.25 mM sodium thiosulphate for 2 hrs in a Dubnoff shaker at 37°C.

even through only a preliminary step of the biotransformation of the drug. The lack of cyanide liberation when succinonltrile is incubated with endoplasmic reticulum "in vitro" and the reported i n e f f e c t i v a e s s

of inducer drugs to increase the

cyanide liberation from succinonitrile 1978)

(Contessa A.R. et al.

strongly suggest that cyanide is the final product of

non inducible steps subsequent to the reactions occurring on the endoplasmic retlculum. The possibility that mitochondrial fraction is involved in succinonitrile biotransformation has been investigated using specific inhibitors of oxidative phosphorylation.

Pharmacological Research Communications, VoL 12, No. 5, 1980 1.

Table

437

The f o r m a t i o n of t h i o c y a n a t e from s u c c i n o n i t r i l e liver slices of rats p r e t r e a t e d w i t h S K F - 5 2 5 A

nMoles

%

thiocyanate/

g/tissue/h -

113.0

SKF-525A

by

variation

16.0

+

38.8 + 12.0

-

65

S K F - 5 2 5 A 35 m g / K g i.p. 45 m i n b e f o r e the sacrifice. I n c u b a t i o n m e d i u m for l i v e r slices as in Fig. I. Results are the m e a n of 5 e x p e r i m e n t s + S.E.

Table

2. The effect

of o x i d a t i v e p h o s p h o r y l a t i o n i n h i b i t o r s on thiocyanate f o r m a t i o n from s u c c i n o n i t r i l e in rat liver slices

nMoles thiocyanate/ g tissue/h

M

Drugs

conc.

-

Rotenone

-6

10 10

-5

119.0

+

4.8

109.0

+

3.3

47.0 + 12.7

Dicyclohexylcarbodiimide

10 -4 -5 10

5.7 +

2,4-Dinitrophenel

10 -4 -3 10 -5 10

118.0

+ 18.3

10 - 4

48.6

+ 12.3

2 x 1 0 ~4

11.3

+

112.0

2.3

+

5.0

89.0 +

2.8

5.1

+

1.2

3.2

I n c u b a t i o n medium for l i v e r slices as in Fig. I. The r e s u l t s are the m e a n of 4 e x p e r i m e n t s + S.E. Table

2 shows

that rotenone,

transport

between

NADH and CoQ,

inhibitor

of e n e r g y

transfer,

an i n h i b i t o r

of e l e c t r o n

dicyclohexylcarbodilmide,

and 2 , 4 - d i n i t r o p h e n o l ,

an

an

Pharmacological Research Communications, VoL 12, No. 5, 1980

438

uncoupling agent, reduced the cyanide formation from succinonitrile. Parallel experiments on rhodanese activity demonstrated that the enzyme was not affected by the drugs. Moreover,

appropriated

controls demonstrated that the used concentrations of inhibitors did not affect the microsomal drug metabolizing enzyme system. The possibility of a cellular damage by the inhibitors was excluded by assessing that there was no difference in D-lactate dehydrogenase released from control and treated slices. Therefore,

the decrease of the m e t a b o l i s m of succinonitrile

by mitochondrial inhibitor or uncoupler agents is a clear indication that this fraction is involved in the biotransformation of the drug. Since both endoplasmic reticulum and mitochondria are apparently involved in succinonitrile metabolism,

several

efforts were made to restore the ability to biotransform the drug by combinlng the two fractions. of required known cofactors acetylCoA,

In spite of full complement + (ATP, FMN, NADP , nicotinamide,

different ionic composition)

these efforts have been

completely unsuccessful. In conclusion,

in the multistep m e t a b o l i s m of succinonitrile,

its interaction with the

endoplasmic reticulum system is

clearly the first process. From this step some intermediates are released that produce cyanide after subsequent metabolizing steps. Particular intracellular equilibria difficult to reproduce may explain the requirement for cellular integrity in the succinonitrile metabolism.

REFERENCES

Cavanna R. and Pocchiari F. (1972) Biochem.Pharmac. 21, 2529 Contessa A.R. and Santi R. (1973), Biochem.Pharmac. 22, 827 Contessa A.R., Floreani M., Bonetti A.C. and Santi R. (1978) Biochem.Pharmac. 27, 1135

Pharmacological Research Communications, VoL 12, No. 5, 1980 Contessa A . R . I F l o r e a n i M., Bonetti A.C. and C a r p e n e d o F. (1978a) 7 th I n t e r n a t i o n a l C o n g r e s s of P h a r m a c o l o g y , Abstr. n. 773, Paris, 16-21 July Curry S°M. (1975) Biochem.Pharmac. 2~4, 351 Gillette J.R. and Sasame H.A. (1964) Fedn. Proc. 23, 537 J o h n s o n M.K. (1960), Biochem.J. 7__7, 610 Lodi F., M a r o z z i E., Barbi G. and Maggi C.A. (1973) I! F a r m a c o (Ed. Pratica) 2__8, 105 Rogers P. and Fouts J.R. (1964), J.Pharmac. Exp.Ther. 146, 286

439