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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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Chang, K.C., Laver, B.A., Bell, T.D. and Chai, Il. (1978). Lancet 1, 1132-1133. Renton, K.W., (1981). Biochem. Pharmacol. 30, 2333-2336. Renton, K.W. and Mannering, G.J. (1976). Drug Metab. Disp. 4, 223-231. Mannering, G.J., Renton, K.W., ElAzhary, R. and Deloria, L. (1980). Ann. N.Y. Acad. Sci. 350, 314-331. Renton, K.W. and Mannering, G.J. (1978). Biochem Biophys. Res. Comm. 12, 343-348.
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).
17. Pharmacol. Exp. Therap. 208, 392-398. Sonnenfeld, G., Harned, C-L., Thaniyavarn, S., Huff, T., Mandel, A.D. and Nerland, D.E. (1980). 969-972.
Antimicrobial Agents and Chemother. 17,
18. Harned, C.L., Nerland, D.E. and Sonnenfeld, G., J. Interferon Res. (in press, 1982).
19. Singh, G. and Renton, K.W. (1981). 20.
Molec Pharmacol. 0, 681-684. Baglioni, C. (1979). Cell 17, 255-262.
1261