Vol. 106, No. 4, 1982 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS June 30, 1982 Pages 1256-1261

HOMOGENEOUS INTERFERON FROM E. COLI DEPRESSES HEPATIC CYTOCHROME P-450 AND DRUG BIOTRANSFORMATION

Gurmit Singh and Kenneth W. Renton

Department of Pharmacology Sir Charles Tupper Medical Building, Dalhousie University

Halifax, Nova Scotia, Canada B3H 4H7

and

Nowell Stebbing*

Genentech, Inc. 460 Point San Bruno Boulevard,

South San Francisco, California 94080, U. S. A.

Received April 26, 1982

SUMMARY: A highly purified homogeneous human interferon produced from cloned genes depressed the levels of hepatic cytochrome P-450 and related xenobiotic metabolism. Using another cloned human interferon and several impure preparations of human and mouse interferon, it appears that only interferons with antiviral activity in the mouse depress cytochrome P-450 in that species. This is the first direct evidence that interferon decreases hepatic drug biotransformation and likely explains the depression of drug elimination which occurs during viral infections or following the administration of interferon inducers.

INTRODUCTION: Cytochrome P-450 and related drug biotransformation in the

liver of animals and man is depressed during viral infections or following

the administration of interferon inducing agents (l-4). Renton and

Mannering proposed that depression of this enzyme system was mediated via

interferon but provided no direct evidence for this hypothesis (5). The

expression of human interferons in Eschericha coli from cloned genes using

recombinant DNA methods has provided highly purified homogeneous interferon

preparations (6-8) allowing the direct testing of the hypothesis. In this

study the effect of two of these highly purified preparations of human

interferon (LeIF-AD and LeIF-A) and several impure preparations of both

mouse and human interferon were tested for their ability to depress

cytochrome P-450 and dependent drug biotransformation in the liver.

*Present address: Applied Genetics, Inc., 1892 Oak Terrace Lane, Newbury Park, Calif. 91320.

0006-291X/82/121256-06$01.00/0 Cop.vnghf 0 1982 b.v .-lcadeernrc Press, Inc.

..I II rrghi s oj reproducrwn rn on I’ .forrn reserved. 1256

Vol. 106, No. 4, 1982 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

MATERIALS AND METHODS: The highly purified human interferons LeIF-A and LeIF-AD were prepared from cultures of E. coli using recombinant DNA techniques (6-8). LeIF-AD is a molecular hybrid formed between two of the human leucocyte interferon subtypes (LeIF-A and LeIf-D). LeIF-A and LeIF-AD were both homogeneous on PAGE and had specific activities of 108 and 2 x 108 units/mg protein respectively. The human buffy coat interferon had a specific activity of 106 units/mg protein. Mouse IFN-(x was a crude serum preparation from mice treated with poly rI.rC and mouse IFN-P was obtained from Calbiochem and had a specific activity of 105 units/mg protein. Poly rI.rC (MW:,lOO,OOO) was obtained from Sigma Chemical Co. BALB/cJ and C57BL/6J were obtained from Jackson Labs, Maine.

The interferon preparations were diluted in sterile saline and were administered i.p. After 24 hours the livers were removed and microsomes prepared by differential ultracentrifugation. Microsomal cytochrome P-450 and b5 were determined by difference spectroscopy (9), the N-demethylation of aminopyrine was determined by the formation of formaldehyde (lo), and the hydroxylation of benzo(a)pyrene was measured by the formation of non-polar fluorescent metabolites (11). Each interferon type was tested in a separate experiment and compared to control mice treated with saline at the same time. The results are expressed as percent of the control f SE for that particular experiment. Also in each experiment a group of mice was treated with the interferon inducing agent poly rI.rC.

RESULTS: The administration of 40,000 units of LeIF-AD resulted in a 47

percent loss of cytochrome P-450 and a 41 percent loss of cytochrome b5 in

microsomes prepared from the livers of BALB c/J mice (Table 1). This loss

of cytochrome P-450 was accompanied by a 55 percent decrease in the

N-demethylation of aminopyrine and a 42 percent decrease in the

hydroxylation of benzo(a)pyrene which are typical xenobiotic

biotransformations carried out by this enzyme system. Similar losses in

cytochrome P-450 and drug biotransformation were also observed in C57BL/6J

strain mice treated with LeIF-AD (Table 1). The changes in this enzyme

system were of similar magnitude to those produced by the interferon

inducer poly rI.rC and are equal to the maximum depression which can be

caused by this agent. Similar losses in cytochrome P-450 and related drug

metabolism were also observed in mice treated with 50,000 units of impure

mouse it and ii type interferon (table 1). This contrasted with highly

purified LeIF-A or a crude preparation of human lL type interferon which had

no effect on cytochrome P-450 or drug biotransformation.

DISCUSSION: The depression of cytochrome P-450 caused by the homogeneous

hybrid interferon (LeIF-AD) of very high purity provides the first direct

evidence that interferon can depress the steady state levels of cytochrome

1257

TABL

E 1

The

depr

essio

n of

he

patic

cy

toch

rom

e P-

450

depe

nden

t dr

ug

oxid

atio

n by

in

terfe

ron

prep

arat

ions

.

Trea

tmen

t C

ytoc

hrom

e P-

450

Cyt

ochr

ome

b5

Amin

opyr

ine

Benz

o(a)

pyre

ne

N-d

emet

hyla

se

Hyd

roxy

lase

BA

LB

c/J

LeIF

-AD

(4

0,00

0 un

its)

53.0

f

4.8*

59

.2

f 0.

1"

54.5

l

3.9*

58

.4

f 3.

6*

LeIF

-A

(50,

000

units

) 11

6.7

l 4.

5 11

0.7

f 3.

9 10

7.1

l 5.

7 88

.7

f 2.

5

Hum

an

IFN

-a

(50,

000

units

) 10

7.0

f 2.

5 10

5.1

* 2.

0 10

3.3

l 3.

0 10

5.4

l 7.

6

Mou

se

IFN

-ir

(50,

000

units

) 58

.6

f 2.

2*

74.3

f

9.2*

59

.7

f 2.

1"

77.3

f

14.8

Mou

se

IFN

-6

(50,

000

units

) 59

.1

f 4.

5*

73.9

f

8.5

50.0

f

4.5*

91

.2

f 4.

5

Poly

rI.

rC

(10

mg/

kg)

67.7

f

5.4"

84

.2

f 7.

8 79

.7

f 4.

3*

96.1

f

10.5

C57

BLj6

J

LeIF

-AD

(5

,000

un

its)

79.4

l

7.1

83.6

f

2.8

81.7

f

10.3

95

.5

l 26

.3

LeIF

-AD

(5

0,00

0 un

its)

58.1

f

8.5"

72

.3

f 17

.3

39.2

f

7.3*

52

.3

f 14

.0"

Poly

rI.

rC

(10

mg/

kg)

56.4

l

8.1*

65

.2

f 11

.4"

39.6

l

6.9*

44

.9

l 2.

0*

Valu

es

are

the

mea

n f

S.E.

of

th

e pe

rcen

tage

s of

co

ntro

l va

lues

ob

tain

ed

for

each

tre

atm

ent

grou

p (n

=4).

The

mea

n va

lues

fo

r al

l co

ntro

ls

in

BALB

/cJ

mic

e w

ere:

.c

yt

P-45

0 =

0.71

*

0.04

nm

oles

/mg

prot

ein;

cy

t b5

=

0.24

f

0.01

nm

oles

/mg

prot

ein;

am

inop

yrin

e N

-dem

ethy

lase

=

384

f 28

nm

oles

H

CH

O/m

g pr

otei

n/hr

; be

nzo(

a)py

rene

hy

drox

ylas

e =

5.1

f 0.

6 nm

oles

3-

OH

-BP/

mg

prot

ein/

hr.

The

mea

n va

lues

fo

r al

l co

ntro

ls

in

C57

BL/6

J m

ice

wer

e:

cyt

P-45

0 =

0.48

f

0.02

nm

oles

/mg

prot

ein;

cyt

b5

= 0.

18

f 0.

01

nmol

es/m

g pr

ot;

amin

oyrin

e N

-dem

ethy

lase

=

406

l 33

nm

oles

H

CH

O/m

g pr

otei

n/hr

; be

nzo(

a)py

rene

hy

drox

ylas

e =

4.6

f 0.

3 nm

oles

3-

OH

-BP/

mg

prot

/hr.

* Si

gnifi

cant

ly

diffe

rent

fro

m

corre

spon

ding

co

ntro

l p<

.0

5.

Vol. 106, No. 4, 1982 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

P-450 in the liver and result in a loss in the capacity of the liver to

metabolize drugs, chemicals and carcinogens. Although it is a human

interferon LeIF-AD is remarkable for its pronounced antiviral activity on

mouse cells (8).

The ability of interferon to depress cytochrome P-450 in the mouse was

not universal to all types of interferon. Although impure preparations of

mouse (t and 6 type interferons which have antiviral activity in the mouse

also depressed cytochrome P-450 and drug biotransformation, another highly

purified homogeneous cloned human leukocyte interferon (LeIF-A) and an

impure preparation of human leukocyte interferon prepared from buffy-coat

cells had no depressant effect on cytochrome p-450 or on drug

biotransformation in mouse liver. It has been observed that LeIF-A, like

human buffy-coat interferon preparations, has no antiviral effects in mouse

cells or mice (7,12). Although we have used only a few types of

interferons in these experiments our data are compatible with the idea that

only interferons with antiviral effects against viral infection in the

mouse can depress cytochrome P-450 in that species.

Although Renton and Mannering previously suggested that interferon

might act as a mediator in depressing cytochrome P-450 during viral

infections or following the administration of interferon inducers (4,5)

they were unable to determine if these effects were due to interferon

itself or to another common property of these agents. Other workers

(13-16) have demonstrated that a loss of cytochrome P-450 occurred in the

liver following the stimulation of other host defense mechanisms but it was

unclear if individual factors such as immune enhancement,

reticuloendothelial cell stimulation, interferon production or a

combination of these was involved in the observed effects. Recent studies

by Sonnenfeld et al. (17, 18) h ave demonstrated that the administration of

crude preparations of immune type interferon (IFN-Y type II) depresses drug

biotransformation in the liver. In our laboratory we have recently

reported that Newcastle Disease Virus causes a loss of cytochrome P-450

1259

Vol. 106, No. 4, 1982 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

only in strains of mice which carry an allele for the high production of

interferon in response to that virus (19). Our present results indicate

clearly that interferon can act as a mediator in depressing the steady

state levels of cytochrome P-450 in the liver.

Direct antiviral and antitumor effects of interferons appear to be

mediated by biochemical pathways leading to inhibition of protein synthesis

(20) * These same mechanisms could be the cause of depression of hepatic

cytochrome P-450 metabolism which would then be an inseparable side-effect

of interferon therapy. It might be possible, by recombinant DNA methods,

to devise interferons which lack effects on cytochrome P-450 mediated drug

biotransformation but which retain pharmacologically desirable effects.

However, the effects of interferons on cytochrome P-450 metabolism are

likely to be significant in relation to clinical usage of these materials.

ACKNOWLEDGEMENTS: We thank Mrs. M. Espiritu for excellent technical

assistance. This work was supported by a grant from the Medical Research

Council of Canada.

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Wetzel, R., Perry, L.J., Estell, D.A., Lin, N. Levine, H.L., Slinker, B*, Fields, F., ROSS, M.J. and Shively, J. (1981). J. Interferon Res. 1, 381-390. Week, P.K., Rinderknecht, E., Estell, D.A. and Stebbing, N. (1981). Infect. Immun. (in press). Week, P.K., Apperson, S., Stebbing, N., Gray, P.W., Leung, D., Shepard, H.M. and Goeddel, D.V. Nucleic Acids Res. (9, 6153-6166). Omura, T. and Sato, R. (1964). J. Biol. Chem. 239, 2370-2378. Sladek, N.E. and Mannering, G.J. (1969). Mol. Pharmacol. 2, 174-185. Nebert, D.W. and Gelboin, H.V. (1968). J. Biol. Chem. 243, 6242-6245. Week, P.K., Apperson, S., May, L. and Stebbing, N. (1981). J. Gen. Virol. 57, 233-237. Farquar, D., Loo, T.L., Gutterman, J.U., Hersh, E.M. and Luna, M.A. (1976). Biochem. Pharmacol. 25, 1529-1535. Soyka, L.F., Stephens, C.C. MacPherson, B.R. and Foster, R.S. (1979). Int. J. Immunopharmacol. 1, 101-112.

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15. Mullen, P.W. (1977). 16.

Br. J. Clin. Pharmacoli, 695-698. Barnes, D.W., Morahan, P.S. Loveless, S. and Munson, E.A. J. (1979).

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Antimicrobial Agents and Chemother. 17,

18. Harned, C.L., Nerland, D.E. and Sonnenfeld, G., J. Interferon Res. (in press, 1982).

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1261