Spin trapping of a free radical intermediate formed during microsomal metabolism of hydrazine

Vol.

133,

BIOCHEMICAL

No. 3, 1985

December

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS Pages

3 1, 1985

SPIN

1086-l

091

TRAPPING OF A FREE RADICAL INTERMEDIATE FORMED DURING MICROSOMAL METABOLISM OF HYDRAZINE Noda,* Kohji Ohno, Toshiaki Sendo, Noda, Hiroshi AyakoMisaka, Yohko Kanazawa, Ryu-ichi Isobe, and Masaharu Hirata**

Atsuko

Faculty of Pharmaceutical Sciences, Kyushu University; University of Occupational *Department of Hospital Pharmacy, & Environmental Health, Japan (Sangyo Ika-Daigaku); **Shionogi Research Laboratory, Shionogi and Co. Ltd. Received

September 23, 1985

SUMMARY: A radical formed during oxidative metabolism of hydrazine in rat liver microsomes was spin-trapped with d-phenyl-tbutylnitrone. The trapped species was identified as hydrazine radical by comparison of its ESR parameters and mass spectrum with those of the adduct formed during CuCl2 catalyzed oxidation of The requirement for oxygen and NADPH in the microsomal hydrazine. oxidation and the occurrence of a typical binding spectrum by difference spectroscopy suggest the involvement of the participation of the cytochrome P-450 enzyme system in the formation of hydrazine radical which must be a precursor of diimide during microsomal oxidation of hydrazine. @ 1985 Academic Press, Inc.

Hydrazine clinical

medicine as well

etc., ducing

the the

organic

derivatives

treatment

of

industrial

antioxidants

production

and rocket

and to

cause

irreversible

worth

noting

that

hydrazine

is

metabolism

of

drugs

(5).

suggested are

Subsequent to

responsible

Abbreviations azodicarboxylate; ridyl 1-oxyl.

involve for

to

of In

spite

oxidative physiologically necrosis

Copyright 0 1985 by Academic Press, Inc. All rights of reproduction in any form reserved.

1086

of

intermediately (3),

(6),

of

and (2).

during

iproniazid

metabolism active

their

damage

(4)

hydrazine

or has

intermediates, mutagenicity

: PBN, cY-phenyl-t-butylnitrone; ADC, TEMPOL, 2,2,6,6-tetramethyl-4-hydroxypipe-

0006-291X/85 $1.50

re-

mutagenic

cellular

formed

in

fungicides,

be potentially

as isoniazid

hepatic

used

hypertension

fuels.

(I),

such

are

tuberculosis,

well-known

hydralazine

which

in

simple

are

is

been

as

hydrazines

carcinogenic

the

its in

agents,

utility,

It

and

potassium

and

Vol.

133,

BIOCHEMICAL

No. 3, 1985

carcinogenicity

(7).

possibility

of

as an ultimate

the

BIOPHYSICAL

The present

work

bioactivation

hepatotoxin,

spin-trapping

AND

of mutagen

RESEARCH

was

done

hydrazine

COMMUNICATIONS

to

investigate

on its

free

and carcinogen,

the radical,

using

the

method.

MATERIALS

AND METHODS

Hydrazine sulfate and phenobarbital (Na salt) were purchased from Tokyo Chemical Ind. Ltd. [15Nlhydrazine (95 atom%) was obtained from the British Oxygen Co. Ltd. NADPH and d-phenyl-tbutylnitrone (PBN) were from Oriental Yeast Co. Ltd. Japan and Aldrich Chemical Co., respectively. Liver microsome phenobarbital-pretreated previously reported

and

isolated hepatocyte Wistar male rats protocols (8,9).

preparations were obtained

from by the

The difference spectra of a substrate (hydrazine or potassium azodicarboxylate:ADC) with rat liver microsomal cytochrome P-450 were recorded at room temperature (2Ok2"C) on a Shimadzu MPS-2000 spectrometer. The experiment was performed in the reaction mixture indicated in the legend of Fig. 1. ESR spectra were recorded JES ME-X spectroscope. As kinds of hydrazine oxidation dation and CuCl2 catalyzed the incubation at 37'C for (chemical oxidation), each benzene. The organic layer fate followed by concentration ford the residue, which was matography (TLC) silica gel layer was divided into six chloroform extract of each the same solvent.

at room temperature (20+1'(Z) on JEOL shown in the legend of Fig. 2, two reactions, i.e. the microsomal oxiautoxidation, were performed. After 20 min (microsomal oxidation) or 10 min reaction mixture was extracted with was dried over anhydrous sodium sulunder a stream of nitrogen to afthen developed on a thin-layer chroplate with ether. The silica gel parts after the development, and the part was used for ESR measurement in

Mass spectra were recorded on JEOL JMS D-300 mass spectrometer comprising of a JMA 3500 computer system. The same samples in which radicals were demonstrated by ESR measurement were used for the mass spectrometry.

RESULTS AND DISCUSSION In

liver

obtained fairly

microsomal

from

or

phenobarbital-pretreated

stable

a maximum

suspension

NADPH-dependent

level

of

isolated

rats, difference

448 nm (Fig.

I),

dition

of metyrapone.

The

spectrum

bolite

from

which

can

hydrazine,

in

1087

hydrazine

spectrum

which suggests

form

hepatocytes

was

produced

characterized

inhibited

a complex

with

by

by the

a presence

of the

a

ad-

a meta-

cyto-

Vol. 133, No. 3, 1985

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Fig. 1 Difference spectra producedby interaction of hydrasine and diimide with cytochrane P-450. Bat liver microsanes were suspended in potassium phosphate buffer solution (pH 7.4). 2.8 ml of the suspension (4.6-4.8 mg/ml) was divided into two cuvettes. After recording a flat base-line, 0.1 ml of a substrate solution (hydrazine or ADC) was added at a concentration of 1.0 mM to the sample cuvette and an equal volume of buffer was added to the reference cuvette, and 0.1 ml of NADPH solution (1.0 r&f) was added to both cuvettes. The tracings A, B, C and D (left figure) represent, respectively, the difference spectra of hydrasine recorded 1.5, 2, 3 and 4 min and A, B, C and D (right figure) represent the difference spectra of diimide recorded 1, 3, 3.5 and 4 min after the addition of NADPH to the sample cuvette.

chrome P-450. of hydrazine, via

it

during

(NH=NH) under

can be deduced the microsomal

that

somes were incubated The signal,

ditions

if

adduct with

either

could

benzene

with

however,

occurs

followed

After

the development,

in the mixture

hydrazine

parts,

each of which

There-

as an intermediate

was not

obtained

by purification silica

of which

liver

micro-

and NADPH in the presence under

added.

from the microsomal

the

gas

of hydrazine.

NADPH or PBN were not

be extracted

ADC instead

of decarboxylation.

diimide

oxidation

by using

decomposes to nitrogen

a condition

A ESR signal was observed

PBN.

was obtained

ADC, as is well-known,

diimide

fore,

The same spectrum

comparable

was then extracted 1088

layer with

con-

The PBN-radical incubation

on a TLC silica gel

of

was divided chloroform,

mixture gel plate. into

six

and ESR

Vol.

133,

No. 3, 1985

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

TEMPOL

(b)

Fig. 2 ESR spectra of PBN-adduct of hydrasine radical. The adduct was obtained from benzene extract after rat liver microscmal oxidation(a) and C&12-catalyzed oxidation(b) of hydrasine at 37V. The reaction mixture of (a) consisted of 5.6 ml of microsanal suspension (4.4 mgbl), 2.0 ml of PBN solution (25 mM), 0.2 ml of hydrazine solution (pH 8.3; 1OmM) and 0.2 ml of NAJJPHsolution (1 mM). The reaction mixture of (b) consisted of 5.0 ml of PBN solution (30 mM), 1.0 ml of hydrazine solution (pH 8.3; 24 mM), 0.5 ml of CuCl2 solution (12 pMM) and 10.0 ml of sodium carbonate solution (0.2 M). The spectra were measured in chloroform by using an extract after TLC purification. ESR conditions : microwave power, 3 mW; modulation amplitude, 1 G; sample temperature, 20°C. TEMPOL : 2,2,6,6-tetramethyl-rlhydroxypiperidyl 1-oxyl.

spectrum

measurement

The ESR signal

was obtained

from the bottom (0.50) large adduct

was performed

of

with

that

of PBN should

in the

3rd zone,

(Fig. of

with

ridyl

1-oxyl,

was

were obtained benzalazine, might

splitting

during

Schiff's

the

latter

somal oxidation.

From the

condensation

was provided

chemical

oxidation Thus the

of the

3rd zone

where the Rf value Therefore,

a trace

a pretty

of the PBN-radical to interrupt

constants On the

Almost 5th

base of hydrazine,

by the

2a),

each extract.

the

of PBN-adduct

other

hand,

2,2,6,6-tetramethyl-4-hydroxypipe-

(chloroform).

17.7G

in benzene.

be formed

Wde , the

compound,

extract

PBN seemed not

however,

were aN=16.6G and aR=3.1G in chloroform. aN of a standard

for

PBN itself.

coexist

The hyperfine

ESR measurement.

from the

the TLC plate

was identical excess

only

in chloroform

the

same signals

zone extract was detected,

of hydrazine

with

from PBN through

the

(to

later)

be mentioned

purification 1089

(Rf:

0.83)

which benzalde-

hydrolysis

by TLC enabled

and microus to avoid

Vol.

133,

No. 3, 1985

confusion

on our

radical both

BIOCHEMICAL

the

idation

was

ESR parameters those

of of

is

Treatments

of

chemical cold

to

during

be the

the

PBN-

autox-

PBN (Fig. are

radical

species in

both

the

identical

The

identical autoxidation

formed

the

2b).

almost

copper-accelerated

under

gave

with

same one as

[15N]hydrazine

of

copper-catalyzed

PBN-adduct the

the

separation

in

chemical

micro-

oxidation.

microsomal

ESR signal

the

and

with

that

as

hydrazine.

radical

spectrometry species

tracts

the

from

the

COMMUNICATIONS

208.

microsomal

adduct

oxidations

Mass

peaks

number,

Therefore,

assumed

by

to be spin-trapped

the

the

of hydrazine. somes

able

RESEARCH

benzalazine(C14H12N2)r

fanned frcmhydrazine

also

BIOPHYSICAL

analysis from

same mass

The radical

with

mass-spectral

adduct(C11H18N30) having

AND

from at

of

both

m/z

molecular

was

performed

PBN-adduct.

the

ion

(M+)

of

the

identification

As indicated

oxidations

207 and 208 in

for

gave which

the

Fig.

characteristic

the

adduct

in

latter of

peak

PBN (m/z

of 3,

the

ex-

fragment

ion

corresponds

to

177)

with

INT. 1000

150

160

110

180

198

2i0

Fig. 3 Mass spectra of PBN-adduct of hydrazine the same one used in ESR experiments as shown in Almost the same spectrum was obtained as in the of hydrazine. When [lsN]hydrazine was used as m/z 207 and 208 observed here shifted to m/z 209

1090

210

the

220

230

radical. The sample is the legend of Fig. 2a. case of chemical oxidation a substrate, the peaks at and 210, respectively.

M/Z

Vol.

133,

BIOCHEMICAL

No. 3, 1985

hydrazine

radical,

possively

be due to M+ of

was

used

*NHNH2

as a substrate

oxidations,

the

peaks

These trapped

observations with

RESEARCH

A peak

31).

concomitant

at

PBN.

instead

of

m/z

207 and

hydrazine,

as

binding

the

the of

cold

m/z

COMMUNICATIONS

177 may

When [15Nlhydrazine

hydrazine

in

208 shifted

demonstrated

first

evidence

metabolic

for

the

hydrazine

for

as the

carbon

both

to m/z

probably processes

hydrazine

kinds

of

209 and

the

participates

in

the

the

or

diimide

hepatotoxic,

by hydrazine

of

formation

has

however,

radical in

mediated

the

The formation

as the

substrate

metabolism

species

formation

of

on the

itself,

results,

itself.

which

oxidation

microsomal

radical

species itself,

study

hydrazine

hydrazine

radical

microsomal

radical

such

the

preliminary During

hydrazine

of

and carcinogenic

in

The present

of

metabolism

from

except

(IO).

process

metabolites

one derived

spectra.

as well

been

that

by the

derivatives oxygen

indicate

diimide

supported

difference

hydrazine

thus

PBN is

can be a precursor

the

(m/z

BIOPHYSICAL

respectively.

210,

of

at

AND

already provide

the

microsomal of

active during mutagenic

itself.

REFERENCES 1.

2. 3. 4. 5.

6. 7. 8.

9. 10.

Toth, B. (1980) J. Cancer Res. Clin. Oncol. 97, 97-108. Sendo, T., Noda, A., Ohno, K., Goto, S., and Noda, H. (19841 Chem. Pharm. Bull. 32, 795-796. Iguchi, S., Goromaru, T., Noda, A., Matsuyama, K., and (1977) Chem. Pharm. Bull. 25, 2796-2800. Sogabe, K. Hsu, K-Y., Noda, A., and Iguchi, S. (1980) J. PharmacobioDyn. 3, 620-627. Noda, A., Matsuyama, K., Yen, S-H., Otsuji, N., Iguchi, S., and Noda, H. (1979) Chem. Pharm. Bull. 27, 1938-1941. Noda, A., HSU, K-Y., Noda, H., Yamamoto, Y., and Kurozumi, T. (1983) J. UOEH (Sangyo-Idai Zasshi) 5, 183-190. Noda, A., Ishizawa, M., Ohno, K., Sendo, T., Goto, S., Noda, H and Hirata, M. (1985) Toxicol. Lett. submitted. &do, T., Noda, A., Noda, H., HSU, K-Y., and Yamamoto, Y. (1984) J. UOEH (Sangyo-Idai Zasshi) 6, 249-255. Noda, A., HSU, K-y., ASO, Y., Matsuyama, K., and Iguchi, S. (1982) J. Chromatogr. 230, 345-352. Augusto, O., Du Plessis, L.R., and Weingrill, L.V. (1985) Biophys. Res. Commun. 126, 853-858. Biochem.

1091