Vol.64,No.3,

1975

BIOCHEMICAL

TETRAHYDROFURANE LIVER Volker

- AN INHIBITOR

MICROSOMAL

Ullrich,

Department

AND BIOPHYSICAL

Peter

March

P450

Weber and Peter Chemistry,

665 Homburg/Saar,

Received

FOR ETHANOL-INDUCED

CYTOCHROME

of Physiological

RESEARCH COMMUNICATIONS

Wollenberg University

German Federal

of the Saarland

Republic

31,1975

Summary Liver

microsomes

from

microsomes

from male

pretreated

rats.

hanced

after

after

after

all

treatments. with

benzpyrene

inhibited

rats.

nounced

ligand-type

low-spin bition

experiments,

ferent

pattern

liver

Lipophilic

organic

P450 dependent

It is now established hydrocarbons in controls

(2,3,4).

nase activities, cytochrome (6,7,8). species postulated

spectrum

It is still that

by Rubin

that

have investigated

after

system

present

pretreatment

basis

is

a proa new

According

to inhi-

rats

have a dif-

a matter

of discussion

can oxidize

ethanol

(9)

which

of differences a general

808

with

whether

and also

rats

from

that

and monoxygeof microsomal

of other

a microsomal is

(1).

polycyclic

differs

heterogeneity by a variety

cyto-

microsomes

in spectra

induced

and by Comai and Gaylor

ethanol-pretreated

by the

in liver

of animals

induced

now supported

Copyright o 19 75 b-v Academic Press. Inc. All rights of reproduction in any form reserved.

ethanol-pre-

produced

EPR-spectrum.

is

whether

the acti-

and concomitantly

known to be metabolized

P450 (P448)

On the

et al.

from

from male and female

are

monoxygenase

which

activity

P450 species.

we have postulated

P450 (5)

exists

in the

compounds

a cytochrome

blocked

in microsomes tetrahydrofurane

microsomes

the

and even more so tetrahydrofurane

difference

of cytochrome

was en-

inhibited

microsomes

P450 species

with

and benzpyrene-

7-ethoxycoumarin

and naphthoflavone Ethanol

0-dealkylation

optical

cytochrome

for

selectively

phenobarbital

in these

have been compared

and phenobarbital-

activity

Metyrapone

the

Only

rats

controls

treatment.

specifically

treated

chrome

and female

The 0-dealkylation

pretreatment

vity

ethanol-pretreated

(10).

can also

findings cytochrome

by ethanol

as

We therefore, perform

the O-

Vol. 64, No. 3, 1975

dealkylation whether

BIOCHEMICAL

of the this

fluorogenic

activity

phenobarbital

AND BIOPHYSICAL

substrate

was different

and benzpyrene

RESEARCH COMMUNICATIONS

7-ethoxycoumarin

from

that

found

(11)

and if

in controls

so,

or after

induction.

Methods Male

and female

barbital

Sprague-Dawley

was given

days.

i.p.

3,4-Benzpyrene

of 20 mg/kg together

for

with

a standard (12)

The test

was dissolved

two days. sucrose

diet

viously

(Altromin

for

Results

and Discussion

compared

recorded

from

the corresponding

ment

to be 3.2

but similar (2.7

large

x 10V5M)

(8).

from

after

with

became apparent

Pb-treated

rats,

and naphthoflavone

It was interesting be inhibited

expected

that

(13).

(11)

and

50 % in weight for

the O-

the monoxygenase

was significantly

use of the

reac-

different

(Bp)-induced

inhibited

the

rats (Pb)

inhibitor

(1.5

x

pretreatmetyrapone

controls

activity

in microsomes

(7,8-benzoflavone)

the 0-dealkylation

the male

et al.

but

proved

in BP-pretreated not the

females

to be rats

(8).

could

also

not be presented

as

by metyrapone.

The inhibition Ki-values

blocking

with pre-

(Table).

metyrapone

for

together

activity

phenobarbital

the

reported,

specific

for

benzpyrene

Km obtained

about

specific

The Km-value

As already

rather

(30 % v/v)

as described

to Gornall

had decreased

a higher

However,

water

weeks

phenothree

in a dose

has been described

x 10m5M and therefore

to the

differences

from

rats

Km-value

for

i.p.

drinking

according

Sodium

DW-2 spectrophotometer.

of 7-ethoxycoumarin.

was found

used.

body weight

and applied

in the

0-dealkylation

but contained

tion

10w6M)

was determined

of ethanol-pretreated

dealkylation

g) were

oil

was given

in an Aminco

to controls

(150-180

of 80 mg/kg

(30 % w/v) for a period of three 8 ). Microsomes were prepared

7-ethoxycoumarin

were

rats dose

in corn

Ethanol

and protein

spectra

Livers

in a daily

since

values straight

as a consequence

listed lines

in the Table were

not

of different

could

obtained cytochrome

809

in reciprocal P450 species.

plots

as

of rats.

Effect

5 x 1o-6

1 x 1o-5

2 x 1o-5

1 x 1o-3 2 x 1o-3 5 x 1o-3

1 x 1o-2

by

7,8-Benzo-

flavone

% Inhibition

Tetrahydro-

furane

No significant

by

differences

2,5 x 10V6

% Inhibition

1)

2 x 1o-5

pone

min.

Metyra-

Prot.

Ativity

The values

of inhibitors

2 x 1o-(j 4 x lo+ 8 x 1O-6

by

% Inhibition

nMol/mq

Specific

Sex

Pretreatment

Table.

I

+ 0,l

observed

15 + 3

10 + 2

621

4+1

5 + 2,5

3+2

-3 + 1

-3 + 1

52 + 5

50 + 7

45 + 6

30 + 2

0,2

d + 0,Ol

5+2

021

7

4

48 + 10

29f

242

14+

3

0 + 0,5

5+2

2+1

l-+1

051

9

12 + 3

0,05

0-dealkylation

4+1

3+1

021

0+1

321

3+1

221

0 + 0,5

72 + 6,5

60 + 5

39 + 3

29 + 2

2,2f0,3

Id+ 9)‘)

Phenobarbital

(+ SD) of 6-8 rats.

microsomal

means

liver

None (Controls)

represent

on the

,: tl

221

221

1 + 0,5

0+1

90 + 3

79 + 4

42 + 5

12 + 3

021

021

0+1

0+1

,

of 7-ethoxycoumarin

+,

32 + 4

24 + 3

12 + 3

a+2

021

of1

0+1

021

34 + 4

28 + 4

23 + 3

15 + 3

,

after

various

,

79 + 5

65 + 6

27 + 2 36 + 4

Oh

021

0+1

021

1+1

021

021

0+1

+.

pretreatments

$ ;

Vol. 64, No. 3, 1975

BIOCHEMICAL

When looking

for

an 0-dealkylation

ethanol

itself

turned

lecules

with

oxygen

functions

furane

(THF)

proved

to have the

a concentration

out

from

were

males Fig.

in microsomes

activity

Pb- or BP-treated

results

optical

controls

to the gives

with

difference

inhibition spectrum

with

THF, isosbestic

for

this

spectral

Fig.

1.

female respond presents control

Difference rats

with

to 0.01, the rats.

spectra increasing 0.12,

spectrum

0.36, obtained

Both microsomal

of liver

rats.

compounds

0.84 with

Microsomes

811

THF at

from

inhibitor

than

spectrum

obtained

female

those

with

female

pretreatment characteristics in agreement points change

THF

rats.

The

indicates

a

of the

pre-

other

fin-

with

at 342 and 398 nm of 3.6

x 10q4M can

-

from

ethanol-pretreated

of THF. The solid

10e2M THF in microsomes

suspensions

tetrahydro-

no effect

and 8.4 mM THF. The dashed

P450/ml.

mo-

metyrapone.

microsomes

concentrations

of small

by 80 %, whereas,

Wavelength

433

rats,

and specificity.

to this

a similar

and a KS-value

a series

and ethanol-pretreated

altered

Upon titration

are obtained

affinity

of the peak at 413 nm after

Ethanol (9).

the

also

Among those

highest

to the

from male

relation

paration.

tried.

the

in ethanol-treated

but

more sensitive

opposite

magnitude

possible

dings

from

1 represents

higher

were

significantly

just

inhibitor

to be effective,

of 10m2M blocks

was seen in microsomes controls

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

contained

2.5

curves

cor-

line

re-

from

male

nMo1 cytochrome

Vol. 64, No. 3, 1975

BIOCHEMICAL

be calculated. showing 2.50

The EPR-spectrum

the

appearance

and 1.90

be explained

is also

modified

of a new low-spin

respectively

(Fig.

2).

by an interaction

atom in THF at the

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

sixth

signal

in the with

The spectral

of the

free

coordination

of THF

gx and g, values

data

electron

position

presence

tentatively

pair

could

of the

of a special

at

oxygen

cytochrome

P450 species. The presented after this female

ethanol form

data

treatment

is

related

control

study

A similar drug

metabolism in the

2.

Effect

conclusion oxidations

microwave

power

amplitude

60 mW, microwave

to be established

is obvious

in rats

that

(14,15)

was derived

Field

chronic

whether

activity

in

the well-known

can be mainly

of the microsomal very

cytochrome

recently

ethanol

from

a

administration

of liver

microsomes from concurve: microsomes

at -18O'C. Upper Lower curve: 2 x 10m2M THF added. 10 G, modulation frequency

812

ex-

-

of THF on the EPR-spectrum

modulation

of such a species

0-dealkylation

under

ethanol-pretreated female rats taining 25 PM cytochrome P450. parameters:

remains

pattern

Magnrlic

Fig.

the existence

it

Nevertheless,

by differences

on various

It

of rats.

of drug

P450 species.

favor

to the THF-inhibited

rats.

sex differences plained

strongly

9.16

frequency GHz.

EPR

100 KHz,

Vol. 64, No. 3, 1975

to male

BIOCHEMICAL

and female

P450 species ly under

rats

with

(16).

special

AND BIOPHYSICAL

The properties

reference

RESEARCH COMMUNICATIONS

of the various

to ethanol

metabolism

cytochrome are

current-

investigation.

Acknowledgements This

work

was supported

Sonderforschungsbereich

by the

Deutsche

38, Project

Forschungsgemeinschaft,

L.

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