Anaerobic dehalogenation of halothane by reconstituted liver microsomal cytochrome P-450 enzyme system

Vol. 102, No. 1,1981 September

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS Pages 507-512

16, 1981

ANAEROBIC DEHALOGENATION OF HALOTHANE BY RECONSTITUTED LIVER MICROSOMAL CYTOCHROME P-450 Kohyu Fujii:

Nobuo Miki,

Toshihiro

ENZYME SYSTEM'

Sugiyama,

Michio Miyake3

and Yoshihiro Department

* ?;

Mario':

Toshio

Yamano

J-:t

National Cardiovascular Center Research Institute, :k Fujishiro-dai, Suita, 565, Department of Anesthesiology, Medical School of ** Hiroshima University, Minami-ku, Hiroshima, 734, Department of Biochemistry, Medical School of Osaka University, Kita-ku, Osaka, 530, Japan Received

of Biochemistry,

July

27,198l SUMMARY

Cytochrome P-450 from liver microsomes of phenobarbital-treated rabbits catalyzed anaerobic dehalogenation of halothane (2-bromo-2-chloro-l,l,ltrifluoroethane) when combined with NADPH and NADPH-cytochrome P-450 reductase. Cytochromes P-45031 and P-448 from liver microsomes of untreated rabbits were less active. Triton X-100 accelerated the reaction. Unlike anaerobic dehalogenation of halothane in microsomes, the major product was 2-chloro-l,l,l-trifluoroethane and 2-chloro-l,l-difluoroethylene was negligible. These products were not detected under aerobic conditions, and dehalogenation activity was inhibited by carbon monoxide, phenyl isocyanide and metyrapone.

INTRODUCTION Halothane This Volatile rabbits

is

compound

an anesthetic, is

metabolites, (2).

metabolized

which

is

in vivo -__

now widely and excreted

CF3CH2C1 and CF2CHC1, are

With

respect

to these

demonstrated

that

aerobic

dehalogenation

CF3COOH (3),

while

anaerobic

reaction

metabolites,

used in clinical in urine also recent

of halothane produces

anesthesia.

as CF3COOH (1).

expired studies

from

anesthetized

have

in microsomes

produces

CF3CH2C1 and CF2CHC1 (4).

1. This study was supported in part by Grant-in-aid for Encouragement of Young Scientists No. 56770670 and Grant-in-aid for Science Research Grant No. 448299 from the Ministry of Education, Science and Culture of Japan. 2. On leave from Department of Anesthesiology, Medical School of Hiroshima University, Minami-ku, Hiroshima 734, Japan. 3. To whom all correspondence should be addressed. 0006-291X/81/170507-06$01.00/0 507

Copsvrighl 1,; 1981 by Academic Press, Inc. .A II rights of reproduction in any form reserrled.

BIOCHEMICAL

Vol. 102, No. 1,1981

Moreover,

these

pretreatment that

reactions

of animals

halothane

is

AND

BIOPHYSICAL

inhibited

by carbon

monoxide

phenobarbital

(3,

These

observations

function

oxidase

are with

metabolized

by microsomal

metabolites

are

produced

depending

CF2CHCl are

not

detected

under

been presented halothane it

is

on dehalogenation

to NADPH-reduced

suggested

that

of reduced

cytochrome

microsomal

proteins

have been reported. dehalogenation CF3CH2C1 is

(5).

the major

species Covalent

The present is

product

catalyzed

MATERIALS

and enhanced

Evidence

Anaerobic

strongly by cytochrome

as the reconstituted

CF CH Cl and 3 2

of

carbene

and complex

to liver

from halothane

indicate P-450

and

has also

of halothane

of fluoride

indicate

spectrum,

trifluoroethyl

by

system

addition

a difference

binding

release

experiments

as far

is

COMMUNICATIONS

In fact,

(4).

exhibited

and anaerobic

of halothane

tension.

of halothane.

the spectral

(6)

mixed

conditions

microsomes

P-450

4).

upon oxygen

aerobic

RESEARCH

(7)

that

anaerobic

(P-450)

and

system

is concerned.

AND METHODS

NADPH (Oriental Yeast Co.), Triton X-100 (Rohm and Haas Co.), metyrapone (a generous gift from Ciba Geigy, Japan) and halothane (Hohechist, Japan) were used, respectively. Phenyl isocyanide was prepared by the method of Schmidt and Stern (8). All other reagents were of the highest grade commercially available. Rabbit liver microsomes were prepared by the method of Miki et al. (9). Intra muscular injection of phenobarbital was made on rabbits (40 mg per kg) at intervals of 24 hrs for 5 days, and then the rabbits were fasted for 24 hrs. Cytochrome P-450 from phenobarbital-treated rabbit liver microsomes (P-450PB) was purified by the method for purification of cytochromes P-450Bl and P-448 (9, 10) with a slight modification. The Step 1 preparation in 5 mM potassium phosphate, pH 7.4, containing 0.5% Triton X-100 (w/v), 20% glycerol (v/v> and 1 mM EDTA (5 &I PTGE-buffer) was chromatographed on a CM-Sepharose CL-6B column. The eluate with 20 mM PTGEbuffer was passed through a cytochrome b5-coupled Sepharose 6B column and the flowthrough fractions were collected. The purified preparation was homogeneous on SDS-gel electrophoresis and the properties were identical to those of P-450 prepared by Imai and Sato (11). Cytochromes P-450Bl and P-448 from untreated rabbit liver microsomes (P-450Bl and P-448) were purified by the method of Miki et al. (9, 10). NADPH-P-450 reductase was purified by the method of Yasukochi and Masters (12) with a slight modification. The concentration of P-450 was determined by the method of Omura and Sato (13). NADPH-P-450 reductase activity was assayed by measuring NADPH-cytochrome c reductase activity in 0.3 M potassium phosphate buffer, pH 7.7, containing 0.1 mM EDTA and at 30°C (12). For measurement of anaerobic dehalogenation activity, the reaction mixture contained 0.85-1.1 nmole of P-450, 0.3 unit of NADPH-P-450 reductase, 0.25 pmole of NADPH, 28.5 pmoles of halothane, 100 rmoles of potassium phosphate buffer, pH 7.4, and 0.03% Triton X-100 (w/v) in the total volume of 0.5 ml. A 5 ml test tube containing the reaction mixture was filled with nitrogen gas and sealed by a double cap. The reaction was initiated by injection of NADPH into the test tube, and the reaction was

508

BIOCHEMICAL

Vol. 102, No. 1,198l

Fig.

1. Gas chromatogram the reconstituted metabolite was conditions are

of the system measured described

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

metabolite. Anaerobic reaction was made by containing 2.6 nmoles of P-448. The at 40°C using DOP column. Other experimental in MATERIALS AND METHODS.

carried out at 37'C. After the reaction, an aliquot of the gas phase taken by a microsyringe and analyzed by a Shimadzu gas chromatograph, GC-4A. Determination of CF3CH2C1 and CFzCHCl was made using authentic materials.

was

model

RESULTS Identification performed

of metabolites: by the reconstituted

chromatography. were

observed

Under (Fig.

Anaerobic

and metabolites

system,

the chromatographic 11, one of which

dehalogenation

conditions

was identified

509

of halothane were employed,

assayed only

was by gas two peaks

as CF3CH2C1 and the

other

Vol. 102, No. 1,198l

BIOCHEMICAL

Table

components specificity

(A)

I.

Obligatory and the

Obligatory

AND

of

BIOPHYSICAL

RESEARCH COMMUNICATIONS

for the anaerobic the activity for (B)

components a)

dehalogenation P-450 isozymes

Specificity

for

activity (B).

P-450

(A)

isozymes a)

Components

Activity

Complete system b) - P-450PB

5.0 (100%)

P-450PB

5.0

(100%)

0.4

P-450Bl

4.0

( 80%)

P-448

2.8

( 56%)

-NADPH-P-450 reductase

(

P-450

8%)

undetectable

- NADPH

Activity

undetectable

- Triton

X-100

2.4

( 48%)

a) The activity is expressed as nmoles of CF3CH2Cl formed / nmole of P-450 per 30 min, and the concentration of each P-450 isozyme was 2.2 nmoles per ml. The was used. b) P-450PB was

as unreacted

reconstituted used

in

this

halothane.

Even when P-448

system

as described

reconstituted

in

MATERIALS

AND METHODS

system.

The peak of CF2CHCl was negligible

was replaced

CF3CH2C1 and halothane

by either

were

P-450PB

observed,

but

(arrow

or P-450B1,

in Fig.

the peaks

the peak of CF2,CHC1 could

1).

of

not be

detected. Obligatory

components

Table

I,

NADPH

was

NADPH-P-450

activity

required

fashion

as

and saturated by either

P-448

of inhibitors

and metyrapone the

test

that

of the control

less

than

reaction

that under

inhibited

0.3 unit

did

not

per

and

the maximum

X-100.

The activity

and increased

in a linear

at least

nmole

in

up to 4 nmoles

per

of NADPH-P-450

of P-450PB. with

P-448

When P-450PB and P-450Bl

respectively.

the reaction. monoxide,

The extent anaerobic

produce

As shown

the reaction

shown that

As shown in Table

by carbon

with

for

upon the concentration

P-450PB,

activity.

observed air

of P-450PB

on the reaction:

tube was replaced

was also

or P-450B 1, the activity

with

strongly

essential

of P-450PB

dependent at around

of halothane:

of 0.03% Triton

concentration

was also

were

It

donor.

in the presence

was 56% and 80% of that Effect

and P-450

electron

increasing

The activity

was replaced

dehalogenation

upon the concentration

with

reductase

anaerobic

reductase

was attained

was dependent

ml.

for

Vhen nitrogen the activity

of inhibition reaction

II,

in microsomes

isocyanide

gas phase decreased

by carbon

any CF3CH2C1 or CF2CHCl.

510

phenyl

in to 24%

monoxide (4).

The

was

Vol. 102, No. 1,198l

Table

II.

BIOCHEMICAL

Effect

of

AND

inhibitors

BIOPHYSICAL

on the

Conditions

anaerobic

Residual activity

Complete

b)

system

+ Ethanol

(0.02%)

+ Phenyl

isocyanide

(20 TM)') C)

+ Metyrapone

(10

ti)

under

carbon

monoxide

under

aird)

RESEARCH COMMUNICATIONS

d)

dehalogenation Per

a)

activity.

cent residual activity (X)

3.8

100

4.1

108

0.3

8

0.3

8

0.9

24

undetectable

0

a)

The activity is expressed as nmoles of CF3CH2Cl formed / nmole of P-450 per 30 min. b) The concentration of each component is described in MATERIALS AND METHODS, and P-450PB (1.7 nmole/ml) was used in this reconstituted system. c) Phenyl isocyanide and metyrapone were dissolved in ethanol, and the concentration of ethanol in the reaction mixture was 0.02%. d) Nitrogen gas in the sealed test tube was replaced by either carbon monoxide or air. Values in parentheses represent the final concentration of the inhibitors and ethanol.

DISCUSSION The

present

of

halothane

In

fact,

investigation is the

containing that

catalyzed

maximum P-450PB

anaerobic of

indicating is

the

is

associated

to

loss

of

trifluoroethyl

with

the

halothane

with

catalyzed

by

remain

result

of

halothane

the

the

mode

of carbene

present molecule

results, along

or is

when is

be is

of

binding

of

complex since

CF'CHCl

P-450 of

to reduced

elimination the

also

showed

complex

511

As

the

of formation

P-450PB,

reaction

CF2CHC1

is

microsomal

microsomal P-450

activity,

the

exist,

chloride has

but mechanism,

not

produced

component or

it

might

membrane. does

by

reconstituted

for

to

observation

enhanced

the

why

might

the is

specific

other

system

with

However,

reason

system.

reconstituted

(4).

Some

unknown.

formation

with

The

dehalogenation oxidase

microsomes

isozymes.

solved.

function

the

necessarily

P-450

anaerobic

consistent in

P-450Bl

not

these

system with

attained

phenobarbital

to

that mixed

This

P-448

reaction

reconstituted

of

used.

the

problems

by

was

either

indicated

P-450-dependent

activity

rabbits

that

several

by

was

containing

halothane

strongly

dehalogenation

pretreatment system

has

be

due

The

formation

to

reconcile

not

seem

and

bromide

been

which

suggested

from

the (5).

Vol. 102, No. 1,1981

In connection

with

were

after

produced

further.

assumed

problem,

even if

Two possible

elucidation

(14).

of these

the

radical

30 min,

problems

it

exists,

mechanisms

it

is

or one-electron

await

might

in which

However,

must

COMMUNICATIONS

and the reaction

reaction

species

RESEARCH

of CF3CH2Cl per nmole

of halothane,

a two-electron

Moreover,

5 nmoles

the complex,

as an intermediate

halothane.

BIOPHYSICAL

for

dehalogenation

by either

AND

the reaction

intermediate.

anaerobic

formed

this

Therefore,

containing for

BIOCHEMICAL

have been proposed

whether

metabolic

continued

be a chloride

a radical

unknown

of P-450

species

CF3CH2Cl is

reduction

has not

been

identified

further

investigations.

was

of yet.

The

REFERENCES 1. Cohen, E. N., Trudell, J. R., Edmunds, H. N., and Watson, E. (1975) Anesthesiology 43, 392-401. 2. Mukai, S., Morio, M., and Fujii, K. (1977) Anesthesiology 47, 248-251. 3. Karashima, D., Hirokata, Y., Shigematsu, A., and Furukawa, T. (1977) J. Pharmacol. Exptl. Ther. 203, 409-416. 4. Fujii, K., Chikasue, F., and Morio, M. (1980) Proc. 1st Japanese Symp. on Biotransformation and Hepatotoxicity of Anesthetics (Morio, M. ed.) Suppl. 16, pp. 11-14, Hiroshima J. Anesthesia. 5. Nastainczyk, W., and Ullrich, V. (1978) Biochem. Pharmacol. 27, 387-392. 6. Uehleke, H., Hellmer, K. H., and Tabarelli-Poplawski, S. (1973) Naunyn-Schmiedeberg's Arch. Pharmacol. 279, 39-52. 7. Van Dyke, R. A., and Gandolfi, A. J. (1976) Drug Metab. Dispos. 4, 40-44. 8. Schmidt, P., and Stern, D. (1929) Beilsteins Handbuch der Organischen Chemie Vol. XII, p. 191, Julius Springer, Berline. 9. Miki, N., Sugiyama, T., and Yamano, T. (1980) .I. Biochem. 88, 307-316. 10. Miki, N., Sugiyama, T., and Yamano, T. (1980) Microsomes, Drug Oxidations, and Chemical Carcinogenesis (Coon, M. J., Conney, A. H., Estabrook, R. W., Gelboin, H. V., Gillette, J. R., and O'Brien, P. J. eds.) Vol. I, pp. 27-36, Academic Press, New York, 11. Imai, Y., and Sato, R. (1974) Biochem. Biophys. Res. Commun. 60, 8-14. 12. Yasukochi, Y., and Masters, B. S. S. (1976) J. Biol. Chem. 251, 5337-5344. 13. Omura, T., and Sato, R. (1964) J. Biol. Chem. 239, 2379-2385. 14. Goldblum, A., and Loew, G. H. (1980) Chem. Biol. Interactions 32, 83-99.

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