Detection of free radical intermediates during isoniazid and iproniazid metabolism by isolated rat hepatocytes

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Biochemical Phamud oby. Vol. 34. No. 3. pp. 381482.1985. Printed in Great Bduin.

0 1985Pergsmon

Press Ltd.

DETECTION OF FREE RADICAL INTERMEDIATES DURING ISONIAZID AND IPRONIAZID METABOLISM BY ISOLATED RAT HEPATOCYTES.

Emanuele

Albano:

Aldo

Tomasi,

§

Vanio

Vannini’

and Mario

U. Dianzani.

@

0 Istituto

di

Patologia

Generale,

Universita

di

Torino,

CSoRaffaello

5 Istituto

di

Patologia

Generale,

Universita

di

Modena,

Via Campi 287,

The two hydrazine culosis sing

and depressive hepatic

Both

the

between been

derivatives

injury

to

follow

reactive

of

and the

(2). are

highly

hepatocytes

are

reactive

described

(4),

At the

end of a Brucker

by electron

sto

et

al.

and in the

200 D 12/10

e.s.r.

oxidation

(5).

the

treatment

of

evident

when acetyl

ted

PBN are

for

of

The e.s.r. served

acetyl

of

PBN (Table

or

spectra

1).

37’C

for

presence

with

of

tuber-

for

cau-

case

of

splitting

Isoniazid

from

(e.s.r.)

monoxygenase

by the

radicals

use

to

spetroscopy

has

acetyl-Isoniazid

P

the

of

form

spin

stable

(3).

from phenobarbital-induced

30 min with

of

adducts

1 mM Isoniazid,

rats,

as

fproniaeid

or

25 mM PBN (Aldrich-Europe,Bersee,Belgium). were

extracted

as in

(4)

and analyzed

hepatocytes

-14.9,

as described

with

Isoniasid

The nitroxide a

H

was performed

according

by Gever

to Augu-

and Hayes

(6).

and Discussion.

m2.49

isopropyl-hydrazine

for

are

hyperfine

splitting

Iproniazid.

added

instead

and Iproniazid,

Similar of

the

free

constants e.8.r.

parent

radical

are c! - 14.4

spectra

compounds

are to

also

isola-

1). obtained

Cu2t

the

the

-dependent 450 radicals (2).

free

reacts

prepared

was synthetized

isolated

N

in

in

released

and isopropyl-hydrazine

detectable.

and u

(Table

following

of

clearly

Isoniazid

hepatocytes

therapy

spectrometer.

Isopropyl-hydrazine

adducts H u -2.47

Italy.

as responsible

now made possible

resonance

were

PBN-radical

Results Following

Italy.

Modena,

and Methods.

at the

is

which

spin

microsomes

incubation

Metal-catalized

the

consisting

cytochrome

to be

radicals

(PBN),

and incubated

and isopropyl-hydrazine

which

by the

postulated

free

nitrone

and liver

acetyl

using

used

indicated

primarly

group,

then metabolized that

detectable

the

widely

and isopropyl-hydrazine,

Materials

previously

been

pathway,

hydrazinic

Acetyl

as phenyl-butyl

adducts,

Isolated

are

and have

same metabolic

intermediates

agents,

nitroxide

ring

respectively,

The identification trapping

respectively

the

acetylated

and Iproniazid

and Iproniazid

41000

(1).

iso-nicotinic

previously

system

syndromes

compounds

the

Isoniazid

30,10125Torino,

catalized

indicating

from

liver

oxidatin that

the

cell of

preparations both

two systems

381

acetyl

are

also

consistent

with

and isopropyl-hydrazine

produce

the

same type

of

in free

those the

ob-

presence

radicals.

Preliminary communications

382 Table

1: Hyperfine

splitting

activation

constants

or the chemical

of

isolated a Isopropyl-hydrasine

The key role

played

addition Table

is

indicated

2: Effect

hyperfine

liver

splitting

chemical

2.53

15.0

2.49

monoxygenase system in the free in the e.s.r.

incubated

Concerning

on the intensity with

features

of

hydrazine acetyl is

presented

of

signal

consistent

treatment

confirm

structure

the PBN adducts

with

with

part

the hypothesis

groups

of

‘4 C-acetyl

the Project

that

Isoniazid

the radical

the e.s.r.

intensity

(2.7).

signal

produced

X inhibition

presence

37%

5 * 0.5

82%

free

radical

trapped,

2+ Cu -mediated

in the radical

by the National

between

oxidation,

formation.

of hepatic

produced

the spectral or isopropyl-

suggest

that

the

This. interpretation

proteins,

following

Cancer Research

(Maryland

rat

(2).

Foundation

40% “Patologia

are

metabolism.

the analogies

14 C-labelling

of

intermediates

and those produced by acetyl

and isopropyl-hydrazine

M.P.I.

1.6

and Iproniazid

or during

might be involved

the reported

both

of

hcpatocytes

This work has been supported and is

0.1 mM

formed by the two drugs

in isolated

and isopropyl

due to the

units)

18 f

monoxygenase system during

either

intensity

of

acetyl-hydrazine.

lx&I

the chemical

signal

activation

28 2 1.4

” + l-naphtyl-isothiocyanate

by the hepatic

radical

such as SKP 525 A and l-naphtyl-isothiocyanate

0.5 &4

The data here

a

H

14.4

e.8.r.

“+SKF525A

II

oxidation

N

2.49

(arbitrary

,t

(Gauss)

14.9

Treatment

Acetyl-hydrarine

constants

a

by the decrease

microsomes

the biological

14.3

of cytochroms P450 inhibitors

by rat

during

aH 2.47

cytochroms P450 inhibitors

of

produced

and isopropyl-hydrazine.

hepatocytes

N

by the microsomal

the above hydrasines

of acetyl

spectra

e.8.r.

Acetyl-hydrazine

the PBN-adducts

oxidation

for

da Radicali

USA)

Liberi”.

References. 1) H.J.

Zimmerman -in:

2) J.A.

Timbrell,

3) E.G.

Janzen &:

“Hepatotoxicityu

Drug Metabolism “Free

Radicals

Press, 4) E. Albano,

K.A.K.

Reviews,

T.F.

lo,

in Biology”

New York

Lott,

pp. 418-510,

Appleton-Century-Crofts,

New York,

(1978).

125 (1979). (Ed. W.A. Pryor)

~01.4,

pp.

116-128,

Academic

(1980).

Slater.

A. Stier,

M.R.C.

Symons and A.

Tomasi,

Biochem. J.,

204, 693 (1982). 5) 0. Augusto,

P.R.

Ortiz 101,

de Hontellano

and A. Quintanilha,

Biochem. Biophys.

1324 (1981).

6) G.
and K. Haves,

7) B. Testa

and P. Jenner.

J. Org.

Chem., 16,

Drug Metabolism

813 (1949).

Review,

12,

1 (1981).

Res.

Conssun..