Apr 30, 1985 - Benzphetamine,. 2.29. 1.79. 22. 4.28. 2.24. 47%. 1 IWl. The reaction mixture contained ethanol-induced or control rabbit liver microsores (0.25 ...
Vol. 128, No. 2, 1985
AND BIOPHYSICAL RESEARCH COMMUNlCATlONS
BIOCHEMICAL
Pages
April 30, 1985
chng
s. Yang+" , Dennis
'Department
March
Tianyuan
War~g*~',
and PIinor
of Biochemistry, UHDNJ-New Jersey Medical Newark, NJ 07103
2Department Received
R. Koog2,
1007-l
013
J. Coon2
School,
of Biological Chemistry, The University of Michigan Mdical School, Ann Arbor, MI 48109
21,
1955
SUMMARY: The ethanol-induced rabbit liver microsomal cytochrome P-450, P450 13a, has been shown previously to efficiently catalyze the demethylation of X-n1Lf rosodimethylamine (NDMA) with a Km of 2.9 mu. Since the predominant hepatic microsomes from ethanol-treated rabbits is 0.07 mE1, the role 450 t3a in the activation of this carcinogen has been uncertain In the present stu 3 y, antibodies to P-450Lpas were shown to almost completely inhibit NDHA demethylation by the purifie -450 in a reconstituted system as well as the low-Km activity of liver microsomes from control or ethanol-treated rabbits. In contrast, the antibody did not inhibit the highNLMA demethylase activity in the microsomes. These results indicate -450 F13a is the major P-450 responsible for the low-Km ?DDE*lA demethylase activity. 4 n addition, evidence is provided for the existence of a cytcchrome i-ocher&ally similar to P-450u,13a in liver microsores from rats, mice, and guinea pigs that effectively catalyzes the demethylation of MJIIA. 0 1985 Academic Press, Inc. Nitrosamines, metabolic
a group of widely
occurring
carcinogens,
are known to require
for conversion
to their
carcinogenic
and cytotoxic
activation
(1,Z). The activation carbon,
process,
generally
involving
has been shown to be a cytochrome
Nevertheless,
the enzymology of nitrosamine
because
the metabolism
prototp
nitrosamine,
reactions
(7-9). Previously,
P-450~dependent metabolism
of N-nitrosodimethylaine is different
of the ~1 -
reaction
is not clearly
characterized
the induction
(2-6).
understood
(hDMA), a commonly
from many well
we have studiti
the oxygenation
forms
studied
monooxygenase
of a high affinity
*This work was supported by Grant CA-37037 and AN-10339 from the NIP. *ro whom requests for reprints should be addressed. kn leave from Henan Tumor Institute, Zhengzhou, Henan, China . . & NDbiA, N-nitrosodimethylamine; NDDplAd,H-nitrosodtithylamine &bbrevlatrons demethylase; control and ethanol-induced microsomes, hepatic microsomes from untreated and ethanol-treated rabbits, respectively. 0006-291X/85
1007
$1.50
Copyright 0 1985 by Academic Press, Inc. All rights of reproduction in any form reserved.
Vol. 128, No. 2, 1985
microsomal
BIOCHEMICAL
NDMA demethylase
acetone,
isopropanol,
diabetes
(10-14).
activity
and pyrazole, The results
the metabolism
been obtained
recently
reconstituted
systems
ethanol-induced
form,
than
five
other
denitrosation other
(NDMAd),with
that
of a specific
by a study
purified
evidence
of NDMA. Theother
liver
h?MAd
has
the
in
(5). The
(lower Km and higher
however,
the metabolism
hypothesis
isozymes
Vmax)
demethylation
and
may bemoreactivewith
the phenobarbital-induced
in catalyzing
and
of nitrosamines
P-450
catalyzing
isozymes,
for example,
than P-450M3a
in
microsomal
for this
metabolism
rabbit
isozymes
such as fasting
P-450 isozyme which is efficient
of the
purified
rats by ethanol,
mM,in
the enhanced
P-450Ut13a, is much more active
nitrosamines;
active
a Km of 0.07
of NDMA. Direct
with
RESEARCH COMMUNICATIONS
as well as by conditions suggest
is due to the induction
in catalyzing
AND BIOPHYSICAL
P-450LE12 ismore
of N-nitrosomethylaniline
(5). An intriguing the nultiple tion
Km values
in rats,
in liver
problem
at least
in the enzymology of NDMA metabolism
found for liver 3 Km values
microsorfes from control
a single
microsomes.
It
ethanol-induced
to elucidate irrmunochemical
rabbits
Km of 2.9 mP1 which
(5).
is higher
rats between a partially
further
than
NDNAd is due to P-
iSOZyi62
In order to resolve
of P-450 isozynes
study was undertaken
the Km of 0.07 m23 in
discrepancy P-450
using antibodies
in
P-450U,13a, however,
low-Km microsomal
purified
for publication). the role
The purified
A similar
enzyme species.
(Tu and Yang, submitted
to the situa-
Tne lowest Km form is predominant
is not known whether this
450LM3a or other
(2,141, Similar
(0.07, 0.27, and 36.8 ti.1) have been observed
rabbits.
the microsomes of ethanol-treated displays
microsores
is the cause of
in nitrosamine
was observed
in
and microsomes this
problem
and
metabolism,
an
prepared against
P-450~43~.
m a. Hepatic microsores were prepared from control or ethanoltreated adult New Zealand male rabbits (2.0 to 2.5 kg) according to previous procedures (15,161. P-450 3a was purified from ethanol-induced microsomes as described previously (16). %e P-450 preparation was electrophoretically homogeneous with a specific content oLP% .O nmol P-450 per mg protein. Electrophoretically wre IUDPH-P-450 reductase was prepared from rabbit liver microsomes (17). The specific activity of the reductase was 54 units (ursol cytochrome c reduced/min/ny protein). 1008
Vol. 128, No. 2, 1985
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
m The antibody to P-450U113a was produced by inmunization of yearling female sh;ep; the IgG fractions from immune and pre-innnune sera were isolated by ammonium sulfate precipitation and DDAE-celiulose colrrmn chromatography (18). significant crossThe final IgG preparation (anti -3a IgG) did not exhibit reactivity with rabbit P-450 isozymes LFl2, LM3b, Isi13c, LE.14or L1.16. A detailed characterization of this antibody preparation is published elsewhere (18). m assays. NDI~lAd activity was assayed by HCHO formation as described previously (13). Tne reaction mixture contained microsomes (0.2 nmol P-450) or the reconstituted monooxygenase system (0.1 nrnol P-450, 0.63 unit NADPH-P-450 reductase, 7.5~g dilauroylphosphatidylcholine), anti-3a IgG, NDMA, and an NADPH-generating system in a final volume of 0.25 ml. Pre-immune IgG was added to the incubations as a control so that the total IgG content was the same in all tne incubation tubes in the experiment. &Nitrosomethylaniline and knzphetamine demathylase activities were also assayed by ECHO forration (13). RESJU!SANlDl.SXJSSIDN
As shown in Figure 1, anti-3a of P-450L113a in a reconstituted 50% inhibition
I$
effectively
nonooxygenase
inhibited system.
It
could be produced by 0.7 ng of anti-3a IgG pr
nq/nmol
P-450, tne antibody
caused a 93% innioition.
tration
of 5 r&l, about 1.7 times the Km of NXM
the NDpIAd activity is estimatedthata nmol P-450.
At 2
NDXA was used at a concenfor P-450L1,13a. The inhibitory
8 t 0 9”
01
mg Anti
3a-IgG/nmol
mgAntl-3o
P-450
IgG/nmol
p-450
Figure L Inhibition of reconstituted MMA demethylase activity by anti3a I$. The reaction mixture contained 0.1 nnol P-450 0.63 unit IWPH-P450 reductase, 7.5 pg dilauroylphosphatidylcholine, anP3i-4. IgG at the indicated concentration, an IWDPH-generatingsystem, and 5 mMNDIWin a final volume of 0.25 ml. The incubation time was 20 min.
m
2. Inhibition of rabbit microsomalNXW demethylaseactivity by anti3a IgG. The reaction mixture contained ethanol-induced rabbit liver microsomes (0.25 nmol P-450) and 0.2 mMNDMA(M) or 5 ml31 NDMA(o---o) in a final volume of 0.25 ml. Other conditions were similar to those for Figure 1. 1009
Vol. 128, No. 2, 1985
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
11@EJNXHDlICALINHIBITION OF RABBIT MICXXXXAL
!muI
NXD3~ACl'MTIE.S
Without antibody
Substrate NfMi,5m NINA, 200 IlM Nitrosomethyl-
aniline,
1 r&l
Benzphetamine,
1 IWl
With antibody
% Inhibition
Without antibody
With antibody
% Inhibition
0.45 1.56
0.13 1.21
71 22
2.39 2.59
0.24 1.16
90% 54%
0.65 2.29
0.42 1.79
35 22
2.21 4.28
1.61 2.24
27% 47%
The reaction mixture contained ethanol-induced or control rabbit liver microsores (0.25 nmol P450) and different substrates. Anti3a IgG was added at a concentration of 1 mg/nnol w50. other conditions are described in Materials and Hethods. The monooxygenaseactivities (in nmol HCHO/min/nmolP-450)and percent inhibition
action
are shown.
of anti-3a
IgG was also
studied
somes from ethanol-treated
rabbits)
(Figure
IgG/nmol
reactions
2).At
0.2 mg anti-3a
with
ethanol-induced
at NDE1Aconcentrations
P-450,the
450, the antibody substrate
almost
concentrations.
completely
of 0.2 and 5.0 nM
inhibition
with 0.2 and 5.0 mH NDMA, respectively.
was 55 and 47% for
At 1.0 mg anti-3a
inhibited
An NDMA concentration
(micro-
microscmes
IgG/nmol
P-
the NDNAd activity
at both
of 0.2 mE1was selected
because
it should produce 74% of the Vmax of the low-Km NDMAd (Km = 0.07 mM) and only 6% of the V,, indicate
of the demethylase
that
very effective
activity
even though purified in inhibiting
The specificity
with
a Km of 2.9 mM. The results
P-450LE13a has a Km of 2.9 mE1, anti-3a
the microsomal
of the inhibitions
low-Km NDMAd activity. was studied
microsomes and control
microsores
(microsones
different
(Table
When assayed with
IgG/nmol
substrates P-450 inhibited
ethanol-induced
1).
71and
microsomes,
tively,
for these two microsomal
results
are consistent
larger control
portion
with the hypothesis
isozymes in addition
and that,
from untreated
rabbits)
of the control
when 200 mM NDMA was used. that P-450U13a
in ethanol-induced
Tne other 1010
and
was 22 and 54%, respec-
is responsible microsomes
at 200 mM, NDWA is metabolized
to P-450LM3a.
and with
5 mM NDMA, 1 mg anti-3a
The inhibition
preparations
of the NDllAd activity
microsones
with both ethanol-induced
90% of the NDMAd activity
respectively.
IgG is
by other
The for a
than in P-450
forms appeared to produce about
Vol. 128, No. 2, 1985
BIOCHEMICAL
TBBLE&
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
INHIBI!TIONOF NDN ACl'IVITy OF MI-MES RA!E,MICE, ANDGUINEAPIGS BYANM-3a IgG
Source of Micro-s
FFOM
Without antibody
With antibody
1.34 6.73
0.39 0.55
2.28 2.23
0.21 0.65
71
8.45
2.01
76
Control rats Ethanol-treated rats Control mice Control guinea pigs Acetone-treated guinea pigs
% Inhibition 71 92 91
The reaction mixture contained microsomes(0.25 nmol p-450) and 5
rnN FENA. Anti-3a I$ was afMed at a concentration of 1 fr&mol Other conditions are described in Materials and nethods. activities
are shcwnin ml
40% of the NDMAd activity 200 mI3 NJXlA; yet this demethylase
activity
(S), was not inhibited
by
than
inhibited
by
P-450LI.13a
inhibited
microsomes.
respectively
in assays containing
also
in control
catalyzes
(5,15),
3a IgG on the metabolism
of these two compounds is duetothe
activity
of other species
from control raboit activities
IgG was also
effective
(Table
of uninduced
was
of inhibition
of benzphetamine action
of anti-
inhibition
of P-
microsomal
NIXlAd
with microsores
rats were about the same as those seen with
1). The antibody
rouse
and ethanol-
and ethanol-induced
liver
The extents
was
of benzpnetamine
the inhibitory
in inhibiting
(Table II).
and ethanol-treated
microsones
it appear s that
more efficiently
control
the metabolism
and nitrosomethylaniline
450LP13a* Anti-3a
(5,10,13).
demethylase
(Table 1). The metabolism
P-450US:3a
of
P-450 (data not shown). The
The nitrosomethylaniline
by 22 and 47%, respectively, &cause
postulations
is known to be catalyzed
(5).
inhibition
which has a very high NDPWd Km value
IgG at lmg/nmol
35 and 27% by the antibody
induced microsomes, also
by anti-3a
a much higher
substrate
with previous
inducible),
of Wnitrosomethylaniline
p-450Lp12
did not produce
than did 5 mFi NDMA. This suggests
P-450 U.,2 (phenobarbital
metabolism
microsomes when assayed with
concentration
the low-Km form of NIXlAd , in agreement Rabbit
NDMAd
HcHo/mirJnnmlP-450.
in the ethanol-induced
substrate
EL450.
nicrosoms
also
as well 1011
effectively
inhibited
as microscmes from control
the and
Vol. 128, No. 2, 1985
acetone-treated the liver
BIOCHEMICAL
guinea
pigs.
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
The results
indicate
that
NDMA is metabolized
of these species by P-450 species that are immunochemically
in
similar
to
P-450L&*a.
From a comparison antibody
exerted
microsomes that
the results
a more potent
the anti-3a
inhibitory system.
IgG to P-450Lh13a ratio
previously
observed
hydroxylation study that
specific
by anti-3a the inhibitory
action
P-450LZ,13a and does not involve suggested
to mediate
demonstrated role
previously
results
are also
P-450-catalyzed
that
superoxide
with
oxidation
in this
radicals,
reactions
and hydroxyl
in the P-450-catalyzedNDMAdactivity
the
and aniline
IgG is due to its direct oxygen
fact
the
IgG to P-450
consistent
established
in
P-450 (i.e.,
than the anti-3a
It was also
of anti-3a
due to
microsomal
of alcohol
reactive
certain
is probably
of the total
inhibition
IgG (18).
2, itappearsthatthe
on the NDIJAd activity
This
was higher
2). The present
land
action
accounts for only a portion
shown in Figure
minor
in Figures
than in the reconstituted
P-450tM3a
ratio
of
previous action
on
which have been (19).
radicals
It play
(Tu andYang,
was also at most a
submitted
for
publication). Although stituted
P-450LM3a has a Km of 2.9 mM for the NDNAd activity
system, anti-3a
microsomes.
These
NDMAd activity different tuted
IgG effectively
results
in microsomes
According
NDMAd, eventhoughthe different
concept,
K,values
microsomal
crossreacts
with
P-450LM3a
ethanol-induced
anti-3a
NDNAd activity IgG.
it
is
environment
of the Km values
of
are mainly
due to the existence
of
interpretation
this
interpretation
P-450 species
remains to be examined. 1012
activity
multiplicity
is due to another
appe ars to be the major
MlMAd activity,
of this
in the local
An alternative
Although
for the low-Km
membrane or in the reconsti-
differences
contributetothe
multiple
parameter
is in the microsomal
~-450 isozymas in microsomes.
the low-K,
because
and the kinetic
to this
in the membrane may also
the low-Km (0.07 mM) NDHAd in
suggest that P-450U,,3a is responsible
when the cytochrome
system
inhibits
in the recon-
P-450 species
is that which
is not favored responsible
for the
Vol. 128, No. 2. 1985
BIOCHEMICAL
Thepresentwork low-Kmmicrosomal
of rats,
in these rodents acetone,
demonstratesthatanti-3a NDMAdactivity
NDMAd activity
responsible NDMA, ethanol,
of rabbit
liver
mice, and guinea pigs.
pyrazole,
imidazole,
This type of cytochrome for the metabolism acetone,
aniline,
RESEARCH COMMUNICATIONS
IgGeffectively
inhibits
as well as the liver The results
of a P-450 LS013atype cytochrome
isopropanol,
14,16,20-22).
AND BIOPHYSICAL
is believed
of toxicologically carbon tetrachloride,
microsomal
suggest the presence
which is inducible
fasting,
the
and other
by ethanol,
treatments
(lo-
to be the major P-450 species important
compounds such as
and enflurane
(14,18,20-
25). 1. 5:
4. 5. 6.
7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19.
20. 21. 2 24. 25.
Magee,P.N., and Barnes,J.M.(1967) Adv.Cancer Res., J,Q, 163-246. Lai,D.Y., and Acros,J.S. (1980) Life Sci., Z, 2149-2165. Guengerich,F.P., Dannan,G.A., Wright,S.T., Martin,W.V., and Kaminsky,L.S. (1982), Biochemistry, a 6019-6030. Czygan,P., Greim,H., Garro,A.J., Hutterer,F., Schaffner,F., Popper,H., and Cooper,D.Y. (1973) Cancer Res., s 2983-2986. Rosenthal,O., Yang, C.S., Tu,Y.Y, Koop,D.R., and Coon,M.J. (1985) Cancer Res., &, 11401145. . Yang,C.S., Tu,Y.Y., Hong,J., and Patten,C. (1984) in N-NitrosQm QccurreBiological Effects & l&&wince fa &man Cancer (O'Nei.1, I.&, von Borstel, R.C., Miller, C.T., Long, J., and Bartsch, H., eds.) pp. 423428, International Agency for Research on Cancer, Lyon, France. Lai,D.Y., Myers,S.C., Woo,Y.T., Greene,E.J., Friedman,M.A., Argus,M.F., and Arcos,J.C. (1979) Chem-Biol. Interactions, Z 107-126. Mostafa,M.H., Rucnirawat,M., and Weisburger,E.K. (1981) Biochem. Pharmacol., & 2007-2011. Lake,B.G., Minski,M.J., Phillips,J.C., Gangolli,S.D., and Lloyd,A.G. (1976) Life Sci.,U,1599-1606. Peng,R., Tu,Y.Y., and Yang,C.S. (1982), Carcinogenesis & 1457-1461. Tu,Y.Y., Peng,R, Chang,Z.-F., and Yang,C.S. (1983) Ciiem. Biol. Interactions, & 247-260. Lewis,K.F., and Yang,C.S. (1981) Biochem. Biophys. Tu,Y.Y., Sonnenberg,J., Res.Comrfiun., U, 905-912. Tu,Y.Y., and Yang,C.S. (1983) Cancer Res., &, 623-629. Peng,R, Tennant,P., Lorr,N.A., and Yanq,C.S. (1983) Carcinogenesis, 4, 703-708. Morgan,E.T., Koop,D.R, and Coon,M.J. (1982) J. Biol. Chem., m, 1395113957. KOOK, D.R, Morgan, E.T., Tarr, G.E., and Coon, M.J. (1982) J. Biol. Chem., & 8472-8480. French, J.S., and Coon, M.J. (1979) Arch. Biochem. Biophys.J.!& 565-577. Koop, R., Nordblom, G.D., and Coon, M.J. (1984) Arch. Eiochem. Biophys. & 228-238. Ingelman-Sundberg, M., and Johansson, I. (1984) J.Biol.Chem.,a, 64476458. Ingelman-Sundberg, M., and Je)rnvall, H. (1984) Biochem. Biophys. Res. Cam., ;a24, 375-382. Koop, D.R., and Coon, M.J. (1984) Mol. Pharmacol., Z, 494-501. 470-481. Kaul, K.L., and Novak, M.F. (1984) Arch. Biochem. Biophys., U, Harris, R.N., and Anders, M.W. (1981) Drug metab. Dispos., 2, 551-556. iMazze,R.I., Woodruff,R.E., and Heerdt,M.E. (1982) Anestnesiol., a, 5-8. Casazza,J.P., Felver,M.E., and Veech,RL. (1984) J. Biol. Chem., m, 231236. 1013
