Dechlorination of chloroethane with a reconstituted liver microsomal system

BIOCHEMICAL

Vol. 53, No. 3, 1973

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

DECHLORINATION OF CHLOROETHANE WITH A RECONSTITUTED LIVER MICROSOMAL SYSTEM,? A. Jay Gandolfi

and Russell

Department

of Anesthesiology

Mayo Clinic,

Received

A. Van Dyke

Rochester,

Minnesota

May 24,1973

microsomal mixed-function oxidase system has SUMMARY. A reconstituted demonstrated the capacity to dechlorinate 1,1,2-trichloroethane. The activity of the reconstituted system was stimulated by the presence of 100,000 g supernatant, similar to intact microsomes, and had characteristics of previous reconstituted systems with different substrates. Previous halogenated vitro

reports anesthetics

by enzymes

oxygen

by Van Dyke et al.

are

and chlorinated

localized

necessary

and the activity

in the

for

activity,

is inducible

to be the mixed-function endogenous It

compounds

the mixed

cytochrome

P-450

cytochrome

c reductase,

components

produced

liver function

microsomal

that

function

oxidases

terminal

and a lipid

a system

(6). system

system

Because

monoxide

inhibits

volatile -in

NADPH and the

the enzymes

metabolize

that

To clarify in

a soluble

reaction,

are believed

steroids,

liver

drugs,

that fraction

(5).

and

fractions:

oxygen),

NADPH

Recombination

of hydroxylating

substrates,

similar

this

system

three

was capable

the involvement

for

into

activates

the dehalogenation

was examined

microsomal

was separated

oxidase

and endogenous

oxidase

microsomes.

that

that

can be dehalogenated

by phenobarbital,

recently

(the

microsomes

liver

shown

(4).

was reported

compounds,

have

ethanes

carbon

oxidases

containing

foreign

(l-3)

of these drugs,

to that

of intact

of the P-450

reaction,

mixed-

the reconstituted

activity.

METHODS Microsomes.--Male with

0.2% sodium

+Supported National

in part Institutes

Sprague-Dawley

phenobarbital by Research of Health,

Copyright @ I973 by Academic Press, Inc. AN rights of reproduction in any form reserved.

in

rats their

(300-350

drinking

g) were water

(7),

Grants GM-17158 and ES-00743 Public Health Service. 687

pretreated fasted from

the

Vol. 53, No. 3,1973

3lCCHEMlCAL

overnight,

and sacrificed

and stored

as a suspension

The 100,000

g supernatant

of microsomes was used

the

day.

Microsomes

-M sucrose

under

(20 mg protein/ml) fresh

(from

RESEARCH COMMUNICATIONS

were

nitrogen

resulting

similarly

prepared at -2O'C

from

treated

the

rats)

(5).

preparation when

this

in assays.

prepared,

-of Components

assayed,

Specific

activities

Lecithin

c reductase* was used

reductase,

ethane-36

at 37'C

of 15% trichloroacetic

acid

overnight

unit

0.5

the

hours.

was added

MgC12,

0.5 pmol

in a total

flask

was

l,l,Z-trichloro(3).

To stop

excess

dehydrogenase,

The reaction

reaction

and the

to allow

(8).

amounts

(pH 7.4).

to start

units

glucose-6-phosphate

of uniformly-labeled

for

in a hood

2.4 rmol

(6).

obtained.

fraction

in various

buffer

as before

and incubated

uncapped

contained

(10.8pmol)

Cl was added

were

mixtures

g Tris-HCl

and 1 pliter

of protein lipid

were

of Lu et al. and 300-400

of the

0.5

fractions

P-450

in place

and lecithin

of 2 ml of 0.05

oxygenated

of cytochrome

glucose-6-phosphate,

P-450,

and P-450

to the method

per milligram

Incubation.--Incubation

and the

reductase

according

of 3 nmoles

(dilauryl)

NADP, 1 pmol

.--The

and stored

NADPH cytochrome

capped

following

in 0.25

was prepared

Preparation

volume

AND BIOPHYSICAL

the

The mixture reaction,

was allowed

was

0.2 ml to stand

1,1,2-trichloroethane

to

volatilize. Assays. were

quantitated Total

mixture it

--Total

before

were

g for

at 1,000

g.

10 minutes, A 0.2-ml

700 ml dioxane:

100 ml Liquifluor)

the 36 Cl-ion

counts per minute *One unit equals

g napthalene:

of inorganic with (cpm). 1 nmol

inorganic

the precipitated

the supernatant,

of the supernatant 420 ml methanol:

and counted

silver

nitrate.

cytochrome

688

c reduced

as before All

and centrifuging was dissolved

in

600 ml toluene:

in a liquid

36 Cl was determined excess

metabolites

36C1 was determined.

by centrifuging

decanting

aliquot

(146

tating

of free

determined

10 ml of Diotol

The amount

36 Cl-labeled

water-soluble

the amount

Cl metabolites

at 1,000

again

non-volatile,

data

scintillation (2) were

per minute.

counter.

by precipirecorded

in

BIOCHEMICAL

Vol. 53, No. 3, 1973

Materials.--Lecithin (San Diego).

before

(dilauryl)

Uniformly

was obtained

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

was purchased

labeled

from New England

from Cal Biochem

1,1,2-trichloroethane-36C1 Nuclear

(Boston)

(0.05

and diluted

mCi/mmol)

with

carrier

use. RESULTS AND DISCUSSION The purified

P-450,

reconstituted

NADPH cytochrome

the carbon-halogen

in

mixture;

The lack

explained

in

required completely

1).

the

optimum

indicate

necessary isolated

reducing

to NADPH in

completely

dechlorinated

in

the total

36 Cl thanpyhe

Table effect. through

water

from

1, the P-450 after forms

1 shows

reported with respect. than

that

Whether

activity,

that the

NADH could

under

active

age rapidly for

Lu et al.

(6)

substrate

but

of reducing

that

it

NADPH and NADH results

NADH reductase-cytochrome

b5 system

have

of was inferior

NADPH was also

equivalents

NADPH cytochrome

are

specificity.

system,

reaction,

since 1 week.

and reductase

as the source

through

689

the trichloroethane.

nitrogen

P-450

36C1

such as mono-

be used

reconstituted

of both

inorganic

components

different

NADH as a source

the NADH is

of

since

In the dechlorination

the addition

a contaminating

with

and therefore

fragments,

and reductase

of the cytochrome

added

was present.

soluble

at -85'C,

be

lipid

of lipid

the dechlorination

storage

of the cytochrome

much more effective

the specific

that

of

might

not

maximum dechlorination

this

was not or the amount

of non-volatile

equivalents

concentration

is

labile

(6)

on the concentration

of P-450

in Figure

Lu et al.

less

primarily

c reductase

amount

resulting

two forms

depended

to cleave

to depend

the small

is decreased

for

was found

and NADPH cytochrome

lecithin

of radioactivity

Possibly,specific

has the capacity

activity

and di-chloroethenol,

the activity

cytochrome

enzymatic

the presence

As shown

containing

on the lecithin the

The 1,1,2-trichloroethane levels

P-450

system

either

saturated

the higher

The system

of dependency

two ways:

for

(Fig.

system

and lecithin

of cytochrome

the incubation

lecithin.

c reductase,

bond

on the concentration

microsomal

(Table

1).

in an additive c reductase is

undetermined.

or

Vol. 53, No. 3, 1973

BIOCHEMICAL

II 400T p450

AND BIOPHYSICAL

Reductase

.

140

I’

300

RESEARCH COMMUNICATIONS

/

/ 200

/ !’ /

1

400

/ i.

012345

0

25 75 125 nmqles qt . c reaucealmm T I

nmoles

T

I

NADH

0 50

Fig.

1.

microsomal

were

held

0.5

fresh

cytochrome

100 pg lecithin. Results

are average

1 also

monoxide,

in

supporting

in

(----), 25 units

36 Cl-

involvement

of 100,000

of NADPH.

constant

they

are

concentrations c reductase, components

NADPH cytochrome

c reductase,

was present

replicate

in all

samples.

and (---) and

assays. (T = total

portion.) of the reconstituted of cytochrome

g supernatant

from

rats

Stimulation

of both

where

l-week-old

system from

NADH and supernatant

NADPH cytochrome with

50 units

reconstituted on each particular

two graphs the

the sensitivity the

the presence

the

concentrations P-450,

listed

constant.

in

from phenobarbital-treated

the presence

added

held

only

I = inorganic

The addition microsomes

were

of duplicates

indicates

with

the component

NADPH generating

36 Cl metabolite; Table

for

P-450,

Constant

1 nmol

dechlorination

components

cytochrome

50 l(g lecithin. were

influencing

or varied

With nmol

Mmoles

components

constant

i”.; 012345

250

Except

other

indicated. were

Factors

system.

all

graph,

150 JJ9

is

NADPH and NADH.

690

to carbon

P-450. the

preparation

stimulated even

system

greater

of liver

dechlorination

activity

when supernatant

Van Dyke and Wineman

(3)

is also

Vol. 53, No. 3,1973

BIOCHEMICAL

Table

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

1 .--Requirements

for

Dechlorination

Additions

Relative

None

activity*

17 +- 1 100

NADPH NADPH + CO NADH

17 +5 34 +- 11

NADPH + supernatant

118 -+ 15

NADPH + NADH (2.5pmol)

109 +- 3

NADPH + NADH -I- supernatant

149 f

Assayed

as described

1 nmol

in Methods.

cytochrome

reductase,

P-450,

50 units

phosphate

NADH = 2.5 pmol.

0.5

and 2.4

Supernatant

expressed

glucose-6

mol MgC12.

activity

NADPH, and are

shown as average

ments

for

both

units

= 100 ~41 (2 mg protein).

as relative

analyzed

c

NADPH 7 0.5 rmol

glucose-6-phosphate, dehydrogenase,

contained

NADPH cytochrome

and 100 pg lecithin.

NADP, lpmol

*Data

All

17

total

to microsomes of duplicate

+

experi-

36C1 and inorganic

36C1 +- SD.

observed used. with

this

stimulation

Preliminary a soluble

evidence protein

When the amounts lecithin,

of activity presence

causes

in

of each

constant, an increase

indicates

intact

liver

the supernatant

microsomes

factor

is

were

associated

labile. P-450,

NADPH cytochrome

an increase in

(Fig.

the amounts different

691

c reductase,

in the concentration

the dechlorination

of each other

by increasing other

very

when

that

of cytochrome

the presence

caused

supernatant

indicates

and is

and NADPH are

and supernatant saturation

by the

1).

activity

to a point

The additional

stimulation

of NADH and supernatant mechanisms

of both

of stimulation.

in the

NADH of

Vol. 53, No. 3,1973

Thus cytochrome

it

appears

P-450,

dechlorinating NADH is that

are

stimulates

BIOCHEMICAL

that

AND BIOPHYSICAL

the reconstituted

NADPH cytochrome

c reductase,

1,1,2-trichloroethane.

approximately similar

microsomal

to those

the activity

of intact

enzyme system

and lecithin

NADPH is

30% as effective.

RESEARCH COMMUNICATIONS

required

is for

The reconstituted microsomes,

containing

capable

activity system

and 100,000

of while

has characteristics

g liver

supernatant

of both. ACKNOWLEDGMENT

We wish

to acknowledge

the

technical

assistance

of Mrs.

Rita

Nelson.

REFERENCES 1.

Van Dyke,

R. A.,

J. Pharmacol.

Exp.

2.

Van Dyke,

R. A.,

and Chenoweth,

R. A.,

and Wineman,

Ther.,

M. B.,

ljq,

Biochem.

364 (1966). Pharmacol.,

14,

603

(1965). 3.

Van Dyke,

C. G.,

Biochem.

Pharmacol.,

20,

463

Fed.

24,

1172

(1971). 4.

Mason,

H. S.,

North,

J. C.,

and Vanneste,

M.,

Proc.,

(1965). 5.

Lu, A. Y. H.,

and Coon,

M. J.,

6.

Lu, A. Y. H.,

Kuntzman,

R.,

J. Biol. 7.

Daly,

8.

Strobel, Chem.,

Chem.,

247,

J. W., Anal. H. W., 245,

4851

1727

J. Biol.

West,

S.,

Chem.,

243,

1331 (1968).

Jacobson,

M.,

and Conney,

A. H.,

(1972).

Biochem.,

Lu, A. Y. H.,

2,

286 (1970).

Heidema,

(1970).

692

J.,

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M. J.,

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