BIOCHEMICAL
Vol. 74, No. 4, 1977
THE INTERACTION Kathryn
OF VINYL CHLORIDE CYTOCHROME P-450
M. Ivanetich,
Department University Received
November
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
of of
Ingrid
WITH RAT HEPATIC --IN VITRO
Aronson*
and I.
MICROSOMAL
Debra
Katz*
Physiology and Medical Biochemistry Cape Town Medical School, Cape Town, South Africa
4,1976
SUMMARY: In the presence of hepatic microsomes, vinyl chloride produces a 'type I' difference spectrum and stimulates carbon monoxide inhibitable NADPH consumption. A comparison of the binding and Michaelis parameters for the interaction of vinyl chloride with uninduced, phenobarbital and 3-methylcholanthrene induced microsomes indicates that the binding and metabolism of vinyl chloride is catalyzed by more than one type P-450 cytochrome, but predominantly by cytochrome P-450. Metabolites of vinyl chloride from this enzyme system decrease the levels of cytochrome P-450 and microsomal heme, but not cytochrome b5 or NADPH-cytochrome c reductase in vitro. Vinyl
chloride
(CH2=CHCl),
is known to be carcinogenic, The toxic
(1,2).
by metabolites system
of
herein
the
of the
hepatic
results
chloride
--in vitro
hepatic
endoplasmic
of vinyl
chloride
mutagenic
effects vinyl
a widely
of vinyl chloride
of with
chloride
of
cytochrome
reticulum
drug
industrially, --in vivo to be mediated
metabolizing
(3-6). the
P-450
proposed
to a dangerous
the
appear
reticulum
an investigation the
compound
and hepatotoxic
from
endoplasmic
used
We report
interaction
enzyme
to function
enzyme
of vinyl
system
of the
in
activation
the
toxin.
EXPERIMENTAL: Vinyl chloride (Matheson, Ltd., U.S.A.) was introduced directly into all suspensions or solutions by bubbling gaseous vinyl chloride through the sample. The final concentration of vinyl chloride was controlled by the time of bubbling. Vinyl chloride was quantitated on a Beckman GC-M gas chromatograph with a 2 m x 4 mm glass column of 10% Carbowax 20M on 80-100 mesh ChromoInjgction port, column and detector temperatures were .s YE8 , w 70 (Hg) and 120 , respectively (7). Male rats of the Long Evans strain, weighing between 180 g and 250 g, were used in all experiments. The treatment and induction of animals and the isolation of hepatic microsomes were as described earlier (8,9). For all experiments, microsomes were suspended at * Recipients
of C.S.I.R.
post-B.Sc.
Copyright 0 1977 by Academic Press, Inc. All rights of reproduction in any form reserved.
bursaries. 1411
ISSN
0006-291X
BIOCHEMICAL
Vol. 74, No. 4, 1977
AND BIOPHYSICAL
Wavelength
Figure
RESEARCH COMMUNICATIONS
(nm)
1
Difference cholanthrene male rats. HCl buffer,
spectra of vinyl chloride induced (A), uninduced Microsomal concentration, pH 7.4; vinyl chloride
with microsomes from 3-methyl(B) and phenobarbital induced (C) 2 mg protein/ml 0.02 M Trisconcentration, 30 mM; 30°.
a concentration of 2 mg protein/ml 0.02 M Tris-HCl buffer, pH 7.4. For determination of the effect of incubation on the levels of microsomal enzymes, microsomal suspensions containing 0.2 mM EDTA (an inhibitor of microsomal lipid peroxidation) were incubated at 30° in the presence of vinyl chloride and/or NADPH generating system (10). Thunberg cuvettes with side arms were used for most experiments since they permitted the addition of reagents from the NADPH consumption results side arm without loss of vinyl chloride. were in all cases corrected for non-cytochrome P-450 dependent (CO inhibitable) NADPH consumption (11). Other experimental procedures are as described earlier (9,lO). RESULTS:
The binding
was investigated
spectrally
cholanthrene chloride
of vinyl
induced binds
of microsomes
to hepatic
in microsomes
1).
chloride
to cytochrome
with
Hanes P-450
KS and AAmax values
from induced
microsomal
investigated
(Fig.
Table
to cytochrome
and phenobarbital
spectrum
and the
chloride
of the
were
linear
calculated
for
3-methylVinyl
in
of a Type
spectral all
therefrom
--in vitro
rats.
P-450
the production
plots
uninduced, male
cytochrome
P-450
all
types
I difference
binding
of vinyl
types
of microsomes,
are presented
in
1. Vinyl
sumption Induction
chloride --in
vitro
increased by hepatic
by 3-methylcholanthrene
carbon
monoxide
microsomes did
1412
inhibitable
from not
variously
appreciably
NADPH coninduced increase
rats. the
BIOCHEMICAL
Vol. 74, No. 4, 1977
Table
1.
The effects
of
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
inducing
vinyl
chloride a. --in vitro
with
agents
hepatic
on the
interaction
microsomal
cytochrome
1.1
0.4~0.2~
1.7 PB
P-450
v max (run01 NADPH/ Km min/mg mic. (mM) 1 protein)
Cyt P-450 (nmol/mg mic. protein)
Pretreatment of animals
of
0.6~0.2~ - I 523 /
3.2
2.320.2
a Abbreviations used are cyt P-450, total type P-450 cytochromes; mic., microsomal; MC, 3-methylcholanthrene; PB, phenobarbital. b Apparent Vmax values measured at the highest concentration of vinyl chloride achievable by bubbling (30 mM);
apparent
Vmax above
that
obtained
For phenobarbital
induced
from
Hanes
was approximately
Vmax
obtained
the
interaction
barbital (Table
plots) with
induced
uninduced
microsomes,
uninduced
of vinyl
for
however, 5 fold
microsomes.
chloride
microsomes
are
microsomes
(Table
Vmax (calculated
greater
than
the
apparent
The KS and Km values
with
cytochrome
identical
1).
within
P-450
for
in pheno-
experimental
error
1).
The effects with
cytochrome
fully
inhibits
of
inhibitors
P-450
--in vitro
the binding
but
does not
decrease
tion
by vinyl
chloride.
ably
inhibit
the
binding
on the
the
are presented
of vinyl
chloride
enhancement In
interaction
contrast,
of vinyl
in
vinyl
Table
chloride
2.
to cytochrome
of CO sensitive metyrapone
chloride
1413
of
SKF 525A P-450
NADPH consumpdoes
to cytochrome
not
appreciP-450
but
BIOCHEMICAL
Vol. 74, No. 4, 1977
Table
The effects
2.
chloride
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
of inhibitors with
on the
hepatic
interaction
microsomal
of vinyl
cytochrome
P-450
--in vitroafb.
Difference
spectra
lA3B5
AdditionsC
PB
None
Uninduced
0.027+0.003
SKF 525A (100 PM)
-0.009+0.003
used
2.320.7
0.022+0.001
0.030~0.004
a Abbreviations b Values are
2.320.9
0.017+0.002
-0.006+0.004
Metyrapone (2.3 mM)
NADPH consumption (nrnol~~;~j;~ mic. P PB
- A41B)
are mic.,
0.79.3
microsomal;
PB, phenobarbital.
means + S.D.
' To samples and references containing microsomal suspension (2 mg protein/ml 0.02 M Tris-HCl, pH 7.4). Samples also contained 10 mM vinyl chloride and, for NADPH consumption studies, 0.24 mu NADPH. Cytochrome P-450 content, 3.2 + 0.3 nmol/mg mic. protein/ PB; 1.1 t 0.2 nmol/mg mic. protein/uninduced microsomes.
does
inhibit
vinyl
the
enhancement
of
CO sensitive
NADPH consumption
by
chloride. The effects
induced
of incubation
rats
with
vinyl
are presented
in
Table
chrome
of hepatic
or NADH (not
3.
The levels
chrome
P-450
in
levels
with
vinyl
are
not
of cytochrome
of
with
plus
cytochrome
is
1414
variously
P-450,
are not
altered
vinyl
chloride,
either
enzymes cytofollowing NADPH
b5 and NADPH-cyto-
following
incubation
of hepatic
NADPH,but
the
of cyto-
The vinyl P-450
from
of microsomal
of cytochrome
affected
chloride
decreased.
levels
c reductase
The levels are
microsomes
on the
microsomes
shown).
c reductase
microsomes
the
chloride
b-5 and NADPH-cytochrome
incubation
chrome
of hepatic
chloride slight
in
levels mediated
control
decrease (8%) and
Vol.
74,
Table
No.
3.
BIOCHEMICAL
4, 1977
The effects
AND
BIOPHYSICAL
of incubation
on the
levels
induced
rats
in
of hepatic
of vinyl
enzymes
microsomal
from
chloride variously
a,b . I
Cyt P-450 (nmol/mg mic. protein) 0 min 15 min
% Loss CYt P-450
VC
None
-
1.252.08
+
1.022.05
1.232.09 0.922.08
-
1.502.10
1.482.06
+
1.352.05
1.242.02
2.362.05
2.322.06
2
+
2.102.10
1.432.10'
32
+d +e
1.982.10
1.992.16
2.042.03
1.582.03'
+f
2.162.01
2.01+.02
+g
2.202.08
1.982.05
PB
COMMUNICATIONS
the presence
Type of Induction
MC
RESEARCH
% Loss CYt b5
% Loss NADPH-cyt reductase
104
96
10
100
99
1
102
8
106
97 91
103
98
100 -
94
2
‘i
0 22
c
-
7
-
10
-
a Samples contained microsomal suspension (2 mg protein/ml 0.02 M NADPH generating system and 0.2mM EDTA. Tris-HCl, pH 7.4), References contained only microsomes. Incubation was at 30° with shaking for I5 min. b Abbreviations used are VC, vinyl chloride (30 mM); cyt, cytochrome; PB, phenobarbital. mic., microsomal; MC, 3-methylcholanthrene; ' An absorbance peak centered at 420 nm was observed in these samples. d Minus NADPH generating system. e Plus 5 mM reduced glutathione. f CO:0 (80:20; v/v) was bubbled through the microsomal suspension afte 1 the addition of vinyl chloride. g NADH (0.6 mM) replaced the NADPH generating system.
3-methylcholanthrene microsomes
heme is of
(7%)
(31%)
cytochrome
In phenobarbital
.
decreased
but
microsomes
by approximately
P-450.
Reduced
is
striking
induced
microsomes,
30% of the
decrease
glutathione
1415
and carbon
in phenobarbital microsomal in
the
monoxide
levels
Vol. 74, No. 4, 1977
80:20;
(CO:02;
the
levels
BIOCHEMICAL
v/v)
vinyl
P-450
mediated
chloride
binds
of
the
microsomes
variation
in
cytochrome
l),
binds
it
vinyl
induction
(Table
predominant
role
cytochrome
P-448
in
does
The stimulation by vinyl by hepatic ment
binding
microsomal
chrome
chloride
P-450
and not
by metyrapone, (see g.~.,
the
(Table
of
The vinyl
chloride
more
--in the
than
AAmax is
vitro. addition that
vinyl
in
the manner
enzyme
(12).
differently
induced
one type
decreased
increased
P-450
by
by phenobarbital
cytochrome chloride
monoxide
cytochrome
P-450 --in
inhibitable the
P-450
plays
vitro
a
whereas
NADPH consumption
metabolism
--in vitro
microsomes
induced
in
microsomes
of vinyl (10).
The enhance-
comparison suggests
chloride
to uninduced that
the metabo-
is catalyzed predominantly by cyto--in vitro greatly by cytochrome P-448. The inhibition
the
2) supports
biotransformation
this
that
that
1) reflects
an inhibitor 13),
P-450
P-450
the
is
of
not.
(Table
of vinyl
cytochrome
indicates
to
of vinyl
of Vmax in phenobarbital
lism
NADH supports
following
with
Since
appear
microsomes
interactions
cytochrome
appear
would
or 3-methylcholanthrene
tion
would
of carbon
chloride
the
(Fig.1)
KS values
but
the
of
of substrates
induction, 1) it
levels
spectra
chloride.
3-methylcholanthrene
3).
in
to NADPH.
microsomes
binding
(Table
cytochrome
microsomal
decrease
induced
to elucidate
I difference
of the
mediated
the
comparison
to hepatic
(Table
in
microsomal
to hepatic
characteristic In view
hepatic
of type
chloride
decrease
attempted
with
The appearance
chloride
chloride
in phenobarbital
30% in
We have
vinyl
vinyl
30% and 80%, respectively
chloride
DISCUSSION:
of
P-450
by approximately
vinyl
the
of cytochrome
by approximately the
inhibit
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
of cytochrome vinyl
chloride
an essential of vinyl mediated
P-450
stimulation role
chloride decrease
1416
dependent
for --in in
reactions
of NADPH consumpcytochrome
P-450
in
vitro. the
levels
of
cytochrome
BIOCHEMICAL
Vol. 74, No. 4, 1977
P-450
in hepatic
to represent
microsomes
in part
from
(ca.
P-450
and in part
cytochrome
P-420
(Table
decrease
partially
fulfilled
carbon
activated
resultant
levels
but
by the
cytochrome
P-450
P-450
the P-450
mediated
decrease
reactive
metabolites
in
conclusion
via
3),
decrease
but
not
(ca.
30%) by glutathione
it
would
proposal
chloride
extensively
but
in in
the
not appear
by cytochrome
This
vinyl
the
decrease
in phenobarbital
P-448.
by
and that
the
metabolism
that
--in vitro these
ence
of hepatic
P-450,
is
is metabolized
by cytochrome
microsomes
effects
of vinyl
macromolecules
The demonstration
P-448
-in
P-450
microsomal
cytochrome
enzyme
in the
the
cytochrome
vinyl
P-450
chloride
suggests
that
such as chloroethylene
decrease
in
the
is considerable
levels
evidence
produced
from
vinyl
and that
they
mediate
chloride
probably
metabolites
system
P-450 levels
of the
chloride
oxide of
cytochrome
suggesting in
the
carcinogenic
by binding
presand
covalently
(4,5,14-17).
that
cytochrome
decrease
are
of
chloride
There
metabolites
to cellular
levels
mediate
(14).
that
mutagenic
the
of vinyl
or chloroacetaldehyde
the
activated
process
(see above). The inhibition
P-450
observed
(Table
chloride
is metaboli-
the
Since
to
NADPH (only
of this
system
microsomes
by cytochrome
with
by cytochrome vitro
is
for
chloride
--in vitro.
is enhanced
induced
extensively
consistent
P-450
P-450
of
P-450
vinyl
enzyme for
appears
heme moiety
of the
vinyl
responsible
rats
of cytochrome
inhibition
that
chloride
the
of cytochrome
3) suggest
cytochrome
vinyl not
levels
are
of
conversion
(Table
3-methylcholanthrene that
the
of cytochrome
of
induced
The requirement
3).
metabolites
levels
the
by NADH) and the
monoxide
cally
the
in
phenobarbital
30%) destruction
cytochrome
mediated
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
mediate
of vinyl the
chloride
destruction
from
of hepatic
--in vitro
provides
an explanation
and total
activity
of hepatic
1417
the
for microsomal
Vol. 74, No. 4, 1977
cytochrome chloride tion
P-450 (6).
for
the
BIOCHEMICAL
following
exposure
In addition, observation
to vinyl
chloride
chloride
(3).
protects
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
these that
prior
against
of
laboratory
results exposure the
toxic
animals
may provide of
to vinyl one explana-
laboratory
effects
animals
of vinyl
Acknowledgements: This research was generously supported by grants from the Cancer Research Fund, the Medical Research Council and the University of Cape Town Staff.Research Fund. We wish to acknowledge the technical assistance and co-operation at all times of Mrs. Jean J. Bradshaw and the assistance of Mrs. Julia A. Marsh with the gas liquid chromatograph. REFERENCES: 1. Seymour, R.B. (Dee 1974 - Jan 1975) Australian Plastics and Rubber, 9-11. Evaluation of 2. World Health Organization - IARC Monograph. Carcinogenic Risk of Chemicals to Man (1974) International Agency for Research on Cancer, Lyon, Vol. 7, 291-318. 3. Jaeger, R.J., Reynolds, E.S., Conolly, R.B., Moslen, M.T., Szabo, S., and Murphy, S.D. (1974) Nature, 252, 724-726. U., Johansson, A., Ramel, C., and Wachtmeister, C.A. 4. Rannug, (1974) Ambio 3, 194-197. H., Malaveille, C., and Montesano, R. (1975) Int. J. 5. Bartsch, Cancer, 15, 429-437. 6. Reynolds, E.S., Moslen, M.T., Szabo, S., and Jaeger, R.J. (1975) Res. Commun. Chem. Path. and Pharmacol., 12, 685-694. 7. Breder, C.V., Dennison, J.L., and Brown, M.E. (1975) J. ASSOC. Off. Anal. Chem., 58, 1214-1220. 8. Ivanetich, K.M., Bradshaw, J.J., Marsh, J.A., Harrison, G.G., and Kaminsky, L.S. (1976) Biochem. Pharmacol., 25, 773-778. 9. Ivanetich, K.M., Bradshaw, J.J., Marsh, J.A., and Kaminsky, L.S. (1976) Biochem. Pharmacol., 25, 779-784. Ivanetich, K.M., Marsh, J.A., Bradshaw, J.J., and Kaminsky, 10. L.S. (1975) Biochem. Pharmacol. 24, 1933-1936, 11. Stripp, B., Zampaglione, N., Hamrick, M., and Gillette, J.R. (1972) Molec. Pharmacol. 8, 189-196. 12. Schenkman, J.B., Cinti, D.L., Moldeus, P.W., and Orrenius, S. (1973) Microsomes and Drug Oxidations, pp. 111-120, Williams and Wilkins Co., Baltimore, Md. 13. Hildebrandt, A.G., and Roots, I. (1973) Nauyn-Schmiedeberg's Arch. Pharmacol., 277, 27-38. 14. Kappus, H., Bolt, H.M., Buchter, A., and Bolt, W. (1975) Nature, 257, 134-135. Barbin, A., Brdsil, H., Croisy, A., Jacquignon, P., Malaveille, 15. C ., Montesano, R., and Bartsch, H. (1975) Biochem. Biophys. Res. Commun., 67, 596-603. 16. Gijthe, R., Calleman, C.J., Ehrenberg, L., and Wachtmeister, C.A. (1974) Ambio, 3, 234-236. 17. Malaveille, C., Bartsch, H., Barbin, A., Camus, A.M., Montesano, R ., Croisy, P. (1975) Biochem. Biophys. A., and Jacquignon, Res. Commun., 63, 363-370.
1418
Vol. 74, No. 4, 1977
THE INTERACTION Kathryn
OF VINYL CHLORIDE CYTOCHROME P-450
M. Ivanetich,
Department University Received
November
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
of of
Ingrid
WITH RAT HEPATIC --IN VITRO
Aronson*
and I.
MICROSOMAL
Debra
Katz*
Physiology and Medical Biochemistry Cape Town Medical School, Cape Town, South Africa
4,1976
SUMMARY: In the presence of hepatic microsomes, vinyl chloride produces a 'type I' difference spectrum and stimulates carbon monoxide inhibitable NADPH consumption. A comparison of the binding and Michaelis parameters for the interaction of vinyl chloride with uninduced, phenobarbital and 3-methylcholanthrene induced microsomes indicates that the binding and metabolism of vinyl chloride is catalyzed by more than one type P-450 cytochrome, but predominantly by cytochrome P-450. Metabolites of vinyl chloride from this enzyme system decrease the levels of cytochrome P-450 and microsomal heme, but not cytochrome b5 or NADPH-cytochrome c reductase in vitro. Vinyl
chloride
(CH2=CHCl),
is known to be carcinogenic, The toxic
(1,2).
by metabolites system
of
herein
the
of the
hepatic
results
chloride
--in vitro
hepatic
endoplasmic
of vinyl
chloride
mutagenic
effects vinyl
a widely
of vinyl chloride
of with
chloride
of
cytochrome
reticulum
drug
industrially, --in vivo to be mediated
metabolizing
(3-6). the
P-450
proposed
to a dangerous
the
appear
reticulum
an investigation the
compound
and hepatotoxic
from
endoplasmic
used
We report
interaction
enzyme
to function
enzyme
of vinyl
system
of the
in
activation
the
toxin.
EXPERIMENTAL: Vinyl chloride (Matheson, Ltd., U.S.A.) was introduced directly into all suspensions or solutions by bubbling gaseous vinyl chloride through the sample. The final concentration of vinyl chloride was controlled by the time of bubbling. Vinyl chloride was quantitated on a Beckman GC-M gas chromatograph with a 2 m x 4 mm glass column of 10% Carbowax 20M on 80-100 mesh ChromoInjgction port, column and detector temperatures were .s YE8 , w 70 (Hg) and 120 , respectively (7). Male rats of the Long Evans strain, weighing between 180 g and 250 g, were used in all experiments. The treatment and induction of animals and the isolation of hepatic microsomes were as described earlier (8,9). For all experiments, microsomes were suspended at * Recipients
of C.S.I.R.
post-B.Sc.
Copyright 0 1977 by Academic Press, Inc. All rights of reproduction in any form reserved.
bursaries. 1411
ISSN
0006-291X
BIOCHEMICAL
Vol. 74, No. 4, 1977
AND BIOPHYSICAL
Wavelength
Figure
RESEARCH COMMUNICATIONS
(nm)
1
Difference cholanthrene male rats. HCl buffer,
spectra of vinyl chloride induced (A), uninduced Microsomal concentration, pH 7.4; vinyl chloride
with microsomes from 3-methyl(B) and phenobarbital induced (C) 2 mg protein/ml 0.02 M Trisconcentration, 30 mM; 30°.
a concentration of 2 mg protein/ml 0.02 M Tris-HCl buffer, pH 7.4. For determination of the effect of incubation on the levels of microsomal enzymes, microsomal suspensions containing 0.2 mM EDTA (an inhibitor of microsomal lipid peroxidation) were incubated at 30° in the presence of vinyl chloride and/or NADPH generating system (10). Thunberg cuvettes with side arms were used for most experiments since they permitted the addition of reagents from the NADPH consumption results side arm without loss of vinyl chloride. were in all cases corrected for non-cytochrome P-450 dependent (CO inhibitable) NADPH consumption (11). Other experimental procedures are as described earlier (9,lO). RESULTS:
The binding
was investigated
spectrally
cholanthrene chloride
of vinyl
induced binds
of microsomes
to hepatic
in microsomes
1).
chloride
to cytochrome
with
Hanes P-450
KS and AAmax values
from induced
microsomal
investigated
(Fig.
Table
to cytochrome
and phenobarbital
spectrum
and the
chloride
of the
were
linear
calculated
for
3-methylVinyl
in
of a Type
spectral all
therefrom
--in vitro
rats.
P-450
the production
plots
uninduced, male
cytochrome
P-450
all
types
I difference
binding
of vinyl
types
of microsomes,
are presented
in
1. Vinyl
sumption Induction
chloride --in
vitro
increased by hepatic
by 3-methylcholanthrene
carbon
monoxide
microsomes did
1412
inhibitable
from not
variously
appreciably
NADPH coninduced increase
rats. the
BIOCHEMICAL
Vol. 74, No. 4, 1977
Table
1.
The effects
of
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
inducing
vinyl
chloride a. --in vitro
with
agents
hepatic
on the
interaction
microsomal
cytochrome
1.1
0.4~0.2~
1.7 PB
P-450
v max (run01 NADPH/ Km min/mg mic. (mM) 1 protein)
Cyt P-450 (nmol/mg mic. protein)
Pretreatment of animals
of
0.6~0.2~ - I 523 /
3.2
2.320.2
a Abbreviations used are cyt P-450, total type P-450 cytochromes; mic., microsomal; MC, 3-methylcholanthrene; PB, phenobarbital. b Apparent Vmax values measured at the highest concentration of vinyl chloride achievable by bubbling (30 mM);
apparent
Vmax above
that
obtained
For phenobarbital
induced
from
Hanes
was approximately
Vmax
obtained
the
interaction
barbital (Table
plots) with
induced
uninduced
microsomes,
uninduced
of vinyl
for
however, 5 fold
microsomes.
chloride
microsomes
are
microsomes
(Table
Vmax (calculated
greater
than
the
apparent
The KS and Km values
with
cytochrome
identical
1).
within
P-450
for
in pheno-
experimental
error
1).
The effects with
cytochrome
fully
inhibits
of
inhibitors
P-450
--in vitro
the binding
but
does not
decrease
tion
by vinyl
chloride.
ably
inhibit
the
binding
on the
the
are presented
of vinyl
chloride
enhancement In
interaction
contrast,
of vinyl
in
vinyl
Table
chloride
2.
to cytochrome
of CO sensitive metyrapone
chloride
1413
of
SKF 525A P-450
NADPH consumpdoes
to cytochrome
not
appreciP-450
but
BIOCHEMICAL
Vol. 74, No. 4, 1977
Table
The effects
2.
chloride
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
of inhibitors with
on the
hepatic
interaction
microsomal
of vinyl
cytochrome
P-450
--in vitroafb.
Difference
spectra
lA3B5
AdditionsC
PB
None
Uninduced
0.027+0.003
SKF 525A (100 PM)
-0.009+0.003
used
2.320.7
0.022+0.001
0.030~0.004
a Abbreviations b Values are
2.320.9
0.017+0.002
-0.006+0.004
Metyrapone (2.3 mM)
NADPH consumption (nrnol~~;~j;~ mic. P PB
- A41B)
are mic.,
0.79.3
microsomal;
PB, phenobarbital.
means + S.D.
' To samples and references containing microsomal suspension (2 mg protein/ml 0.02 M Tris-HCl, pH 7.4). Samples also contained 10 mM vinyl chloride and, for NADPH consumption studies, 0.24 mu NADPH. Cytochrome P-450 content, 3.2 + 0.3 nmol/mg mic. protein/ PB; 1.1 t 0.2 nmol/mg mic. protein/uninduced microsomes.
does
inhibit
vinyl
the
enhancement
of
CO sensitive
NADPH consumption
by
chloride. The effects
induced
of incubation
rats
with
vinyl
are presented
in
Table
chrome
of hepatic
or NADH (not
3.
The levels
chrome
P-450
in
levels
with
vinyl
are
not
of cytochrome
of
with
plus
cytochrome
is
1414
variously
P-450,
are not
altered
vinyl
chloride,
either
enzymes cytofollowing NADPH
b5 and NADPH-cyto-
following
incubation
of hepatic
NADPH,but
the
of cyto-
The vinyl P-450
from
of microsomal
of cytochrome
affected
chloride
decreased.
levels
c reductase
The levels are
microsomes
on the
microsomes
shown).
c reductase
microsomes
the
chloride
b-5 and NADPH-cytochrome
incubation
chrome
of hepatic
chloride slight
in
levels mediated
control
decrease (8%) and
Vol.
74,
Table
No.
3.
BIOCHEMICAL
4, 1977
The effects
AND
BIOPHYSICAL
of incubation
on the
levels
induced
rats
in
of hepatic
of vinyl
enzymes
microsomal
from
chloride variously
a,b . I
Cyt P-450 (nmol/mg mic. protein) 0 min 15 min
% Loss CYt P-450
VC
None
-
1.252.08
+
1.022.05
1.232.09 0.922.08
-
1.502.10
1.482.06
+
1.352.05
1.242.02
2.362.05
2.322.06
2
+
2.102.10
1.432.10'
32
+d +e
1.982.10
1.992.16
2.042.03
1.582.03'
+f
2.162.01
2.01+.02
+g
2.202.08
1.982.05
PB
COMMUNICATIONS
the presence
Type of Induction
MC
RESEARCH
% Loss CYt b5
% Loss NADPH-cyt reductase
104
96
10
100
99
1
102
8
106
97 91
103
98
100 -
94
2
‘i
0 22
c
-
7
-
10
-
a Samples contained microsomal suspension (2 mg protein/ml 0.02 M NADPH generating system and 0.2mM EDTA. Tris-HCl, pH 7.4), References contained only microsomes. Incubation was at 30° with shaking for I5 min. b Abbreviations used are VC, vinyl chloride (30 mM); cyt, cytochrome; PB, phenobarbital. mic., microsomal; MC, 3-methylcholanthrene; ' An absorbance peak centered at 420 nm was observed in these samples. d Minus NADPH generating system. e Plus 5 mM reduced glutathione. f CO:0 (80:20; v/v) was bubbled through the microsomal suspension afte 1 the addition of vinyl chloride. g NADH (0.6 mM) replaced the NADPH generating system.
3-methylcholanthrene microsomes
heme is of
(7%)
(31%)
cytochrome
In phenobarbital
.
decreased
but
microsomes
by approximately
P-450.
Reduced
is
striking
induced
microsomes,
30% of the
decrease
glutathione
1415
and carbon
in phenobarbital microsomal in
the
monoxide
levels
Vol. 74, No. 4, 1977
80:20;
(CO:02;
the
levels
BIOCHEMICAL
v/v)
vinyl
P-450
mediated
chloride
binds
of
the
microsomes
variation
in
cytochrome
l),
binds
it
vinyl
induction
(Table
predominant
role
cytochrome
P-448
in
does
The stimulation by vinyl by hepatic ment
binding
microsomal
chrome
chloride
P-450
and not
by metyrapone, (see g.~.,
the
(Table
of
The vinyl
chloride
more
--in the
than
AAmax is
vitro. addition that
vinyl
in
the manner
enzyme
(12).
differently
induced
one type
decreased
increased
P-450
by
by phenobarbital
cytochrome chloride
monoxide
cytochrome
P-450 --in
inhibitable the
P-450
plays
vitro
a
whereas
NADPH consumption
metabolism
--in vitro
microsomes
induced
in
microsomes
of vinyl (10).
The enhance-
comparison suggests
chloride
to uninduced that
the metabo-
is catalyzed predominantly by cyto--in vitro greatly by cytochrome P-448. The inhibition
the
2) supports
biotransformation
this
that
that
1) reflects
an inhibitor 13),
P-450
P-450
the
is
of
not.
(Table
of vinyl
cytochrome
indicates
to
of vinyl
of Vmax in phenobarbital
lism
NADH supports
following
with
Since
appear
microsomes
interactions
cytochrome
appear
would
or 3-methylcholanthrene
tion
would
of carbon
chloride
the
(Fig.1)
KS values
but
the
of
of substrates
induction, 1) it
levels
spectra
chloride.
3-methylcholanthrene
3).
in
to NADPH.
microsomes
binding
(Table
cytochrome
microsomal
decrease
induced
to elucidate
I difference
of the
mediated
the
comparison
to hepatic
(Table
in
microsomal
to hepatic
characteristic In view
hepatic
of type
chloride
decrease
attempted
with
The appearance
chloride
chloride
in phenobarbital
30% in
We have
vinyl
vinyl
30% and 80%, respectively
chloride
DISCUSSION:
of
P-450
by approximately
vinyl
the
of cytochrome
by approximately the
inhibit
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
of cytochrome vinyl
chloride
an essential of vinyl mediated
P-450
stimulation role
chloride decrease
1416
dependent
for --in in
reactions
of NADPH consumpcytochrome
P-450
in
vitro. the
levels
of
cytochrome
BIOCHEMICAL
Vol. 74, No. 4, 1977
P-450
in hepatic
to represent
microsomes
in part
from
(ca.
P-450
and in part
cytochrome
P-420
(Table
decrease
partially
fulfilled
carbon
activated
resultant
levels
but
by the
cytochrome
P-450
P-450
the P-450
mediated
decrease
reactive
metabolites
in
conclusion
via
3),
decrease
but
not
(ca.
30%) by glutathione
it
would
proposal
chloride
extensively
but
in in
the
not appear
by cytochrome
This
vinyl
the
decrease
in phenobarbital
P-448.
by
and that
the
metabolism
that
--in vitro these
ence
of hepatic
P-450,
is
is metabolized
by cytochrome
microsomes
effects
of vinyl
macromolecules
The demonstration
P-448
-in
P-450
microsomal
cytochrome
enzyme
in the
the
cytochrome
vinyl
P-450
chloride
suggests
that
such as chloroethylene
decrease
in
the
is considerable
levels
evidence
produced
from
vinyl
and that
they
mediate
chloride
probably
metabolites
system
P-450 levels
of the
chloride
oxide of
cytochrome
suggesting in
the
carcinogenic
by binding
presand
covalently
(4,5,14-17).
that
cytochrome
decrease
are
of
chloride
There
metabolites
to cellular
levels
mediate
(14).
that
mutagenic
the
of vinyl
or chloroacetaldehyde
the
activated
process
(see above). The inhibition
P-450
observed
(Table
chloride
is metaboli-
the
Since
to
NADPH (only
of this
system
microsomes
by cytochrome
with
by cytochrome vitro
is
for
chloride
--in vitro.
is enhanced
induced
extensively
consistent
P-450
P-450
of
P-450
vinyl
enzyme for
appears
heme moiety
of the
vinyl
responsible
rats
of cytochrome
inhibition
that
chloride
the
of cytochrome
3) suggest
cytochrome
vinyl not
levels
are
of
conversion
(Table
3-methylcholanthrene that
the
of cytochrome
of
induced
The requirement
3).
metabolites
levels
the
by NADH) and the
monoxide
cally
the
in
phenobarbital
30%) destruction
cytochrome
mediated
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
mediate
of vinyl the
chloride
destruction
from
of hepatic
--in vitro
provides
an explanation
and total
activity
of hepatic
1417
the
for microsomal
Vol. 74, No. 4, 1977
cytochrome chloride tion
P-450 (6).
for
the
BIOCHEMICAL
following
exposure
In addition, observation
to vinyl
chloride
chloride
(3).
protects
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
these that
prior
against
of
laboratory
results exposure the
toxic
animals
may provide of
to vinyl one explana-
laboratory
effects
animals
of vinyl
Acknowledgements: This research was generously supported by grants from the Cancer Research Fund, the Medical Research Council and the University of Cape Town Staff.Research Fund. We wish to acknowledge the technical assistance and co-operation at all times of Mrs. Jean J. Bradshaw and the assistance of Mrs. Julia A. Marsh with the gas liquid chromatograph. REFERENCES: 1. Seymour, R.B. (Dee 1974 - Jan 1975) Australian Plastics and Rubber, 9-11. Evaluation of 2. World Health Organization - IARC Monograph. Carcinogenic Risk of Chemicals to Man (1974) International Agency for Research on Cancer, Lyon, Vol. 7, 291-318. 3. Jaeger, R.J., Reynolds, E.S., Conolly, R.B., Moslen, M.T., Szabo, S., and Murphy, S.D. (1974) Nature, 252, 724-726. U., Johansson, A., Ramel, C., and Wachtmeister, C.A. 4. Rannug, (1974) Ambio 3, 194-197. H., Malaveille, C., and Montesano, R. (1975) Int. J. 5. Bartsch, Cancer, 15, 429-437. 6. Reynolds, E.S., Moslen, M.T., Szabo, S., and Jaeger, R.J. (1975) Res. Commun. Chem. Path. and Pharmacol., 12, 685-694. 7. Breder, C.V., Dennison, J.L., and Brown, M.E. (1975) J. ASSOC. Off. Anal. Chem., 58, 1214-1220. 8. Ivanetich, K.M., Bradshaw, J.J., Marsh, J.A., Harrison, G.G., and Kaminsky, L.S. (1976) Biochem. Pharmacol., 25, 773-778. 9. Ivanetich, K.M., Bradshaw, J.J., Marsh, J.A., and Kaminsky, L.S. (1976) Biochem. Pharmacol., 25, 779-784. Ivanetich, K.M., Marsh, J.A., Bradshaw, J.J., and Kaminsky, 10. L.S. (1975) Biochem. Pharmacol. 24, 1933-1936, 11. Stripp, B., Zampaglione, N., Hamrick, M., and Gillette, J.R. (1972) Molec. Pharmacol. 8, 189-196. 12. Schenkman, J.B., Cinti, D.L., Moldeus, P.W., and Orrenius, S. (1973) Microsomes and Drug Oxidations, pp. 111-120, Williams and Wilkins Co., Baltimore, Md. 13. Hildebrandt, A.G., and Roots, I. (1973) Nauyn-Schmiedeberg's Arch. Pharmacol., 277, 27-38. 14. Kappus, H., Bolt, H.M., Buchter, A., and Bolt, W. (1975) Nature, 257, 134-135. Barbin, A., Brdsil, H., Croisy, A., Jacquignon, P., Malaveille, 15. C ., Montesano, R., and Bartsch, H. (1975) Biochem. Biophys. Res. Commun., 67, 596-603. 16. Gijthe, R., Calleman, C.J., Ehrenberg, L., and Wachtmeister, C.A. (1974) Ambio, 3, 234-236. 17. Malaveille, C., Bartsch, H., Barbin, A., Camus, A.M., Montesano, R ., Croisy, P. (1975) Biochem. Biophys. A., and Jacquignon, Res. Commun., 63, 363-370.
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