- Email: [email protected]
Formation and reduction of a nitroxide radical by liver microsomes
Biochemical
Pharmacology,
Pergsmon
Vol. 26, pp. 675-678.
FORMATION
Printed
Press, 1977.
AND REDUCTION
in Great
OF A NITROXIDE
Britain
RADICAL
BY LIVER MICROSOMES
Gerald M. Rosen and Elmer J. Rauckman Department
of Physiology
and Pharmacology, Durham,
both a reducing non-specific reduction
P-450
is termed
enzyme
system
of molecular
act as a reducing substrate
replaces
Stable
(2).
low reactivity
Stier and Reitz
(3) have reported
reduced
in the presence
that (I) could methylpiperidine
(II)
stable equilibrium (e.p.r.)
spectroscopy,
reduction decided
to reinvestigate
The synthesis method
of Rauckman
chloroperbenzoic
__. et al
acid.
that an equilibrium
Reduction
For the drug induction
(III)
the
since they are paramagnetic,
and have a terminal
oxygen.
(I) (Fig. 1) is
They also reported oxidation
by electron
(4)
of 2,2,6,&tetra-
and oxyqen,
and that a
paramagnetic
the oxidation
resonance
of (II)
(3,4) gave no experimental
(I) was carried
and the
details,
out according
of 2,2,6,6_tetramethylpiperidine oxide and hydrogen
as reported solution
in the literature
(7).
we
to the
using E-
(II)
gave l-hydroxy-
The microsomes
such that a final concentration
The microsomal
of Lowry etz.(8)
protein
The liver microsomes
were isolated
(9)
were
of 0.5 g of
and cytochrome
and flmura and Sato
, rats were injected intraperitoneally
each day for 4 days.
to be reduced,
(6).
chloride
by the methods
P-450 is to
sequences.
was obtained.
studies
in which
enzymes
between
of (I) with platinum
were prepared
of suspension
tents were determined
(80 mg/kg)
of the microsomal
(5) by oxidation
in 1.15% potassium
liver/ml
by the NADPH-dependent
these reaction
other than the
if cytochrome
and NADPH.
of 2,2,6,6_tetramethylpiperidinoxyl
Liver microsomes
whole
oxygen
Since Stier and Reitz
2,2,6,6_tetramethylpiperidine
resuspended
conditions
of (I) could be observed
indicating
of (I) is achieved.
processes
that 2,2,6,6-tetramethylpiperidinoxyl
in the presence
concentration
in reducing
as oxygen analogs
under physiological
enzymatically
of
with the ferrohemochrome.
of liver microsomes,
be formed
the presence
nature of this relatively
intermediate,
can be considered
have relatively
Center,
1977)
since it requires
form of the cytochrome
in its reaction
radicals
31 January
The ambivalent
(1).
The reductive
is the ferrous
oxygen
nitroxide
oxygen
oxidase
has led to its implication
oxygen
agent,
1976; accepted
a mixed-function
agent and molecular
Medical
N.C. 2771n, U.S.A.
(Received 28 October
Cytochrome
Duke University
P-450 con-
respectively.
with phenobarbital as reported
earlier.
616 Electron
Preliminary Communications resonance spectra were obtained
Paramagnetic
E-9 spectrometer.
The kinetic
in the height of the central typical
(III),
experiment,
studies
were conducted
peak of the nitroxide
the reaction
medium
by measuring
triplet
contained
5 x 10
2.5 x 1o-4 M of NADPH, 0.1 ml of the microsomal
solution
contained
the final volume
using a Varian
-5
the decrease
as a function
M of the nitroxide
enzymes
These
experiments
were conducted
model
or increase
of time.
In a
or hydroxylamine
and sufficient
2.33 x 10 -2 M KH2P04 and 8.33 x low3 M MgC12 adjusted
to 0.5 ml.
Associates
buffer
(the
to pH 7.4) to bring
at ambient
temperature
in a
0.1 rnn flat cell. 2,2,6,6_Tetramethylpiperidine ambient
temperature
in the presence
used: the first containing except the microsomal the concentration Incubating oxygen
(1 x 1De4 M) was incubated
everything
enzymes.
solution
of the nitroxide
in the e.p.r.
oxygen was bubbled
(II)
of the amine
of 1 hr.
(II)
of the amine
to the nitroxide
with the microsomal
enzymes
These
observation
aliquots
experiments
suggested
(I) via (III)
of (II)
to (I).
in the presence
of air (lo), suggesting step in the conversion
nitroxide
formation
from the amine
aerobic
conditions
We feel that the enzyme
that cytochrome
oxidation
is not increased
inhibited
by SKF-525A
monoxide
min
by Ziegler
-1
by specific
responsible
and Mitchell
is not responsible
using phenobarbital-induced
led to the formation
measurable
pmole -' of cytochrome antagonists
amount of dithionite
e.p.r.
signal. min
-1
pmole
Evidence
for this oxidation: microsomes,
inhibitors
This reaction -1
for this oxidation (11).
of the
of is the
was gathered
(1) the rate of
(2) the oxidation
of cytochrome
undergoes
is not
P-450, and (3) carbon
by 50 per cent when added to dithionite-reduced
that the nitroxide
(III) at a kinetically
8.9 x 1o-3 m-moles
with a minimal
P-450
this reaction
We have also observed
competitively
described
as well as other specific
only inhibited
hydroxylamine
of its characteristic
of (III)
(II), incubation
to have a Km of 3.2 x 10 -4 M and V,,, of 8.4 x 1O-3 m-moles
to demonstrate
the
On the other hand, it is known
was found
amine oxidase
that either
(I).
by the appearance
mixed-function
through which
or that the rate of reduction
could be the rate-limiting
and NADPH under
(Fig. 2).
enzymes,
of an oxygen-saturated
from a mixture
as evidenced
P-450
and
(I) over that of the
nitroxide
cytochrome
were
spectrometer
of the microsomal
of the nitroxide
rapid autoxidation
we did not observe
at
everything
in an e.p.r.
in the presence
both by continuous
to the nitroxide
relatively
oxidation
Althouqh
(II)
than the rate of oxidation
(II)
were then placed
the concentration
a period
Two controls
and the second containing
cell for 30 min and by taking
during
undergoes
that enzymatic
NADPH,
enzymes
was determined.
was performed
is not oxidized
of (I) is greater that (III)
except
2,2,6,6_tetramethylpiperidine
This experiment
piperidine
(2.5 x 10e4 M) and oxygen.
These mixtures
and NADPH did not increase
control.
of NADPH
with the microsomal
an NADPH-dependent
reduction
microsomes. to the
rate with a Km of 1.8 x low4 M and a Vmax of P-450
of cytochrome
(Fig. 3). P-450.
and then exposing
This reduction
Pretreating
the enzymes
is inhibited
the microsomal
to carbon monoxide
enzymes prior
Preliminary
to introducing
the nitroxide
other hand, this reduction
and NADPH prevented
was greatly
were used in place of control
61-l
Communications
increased
the reduction
of the free radical.
if phenobarbital-induced
On the
microsomal
enzymes
enzymes.
-0 H
47
II
III
Y
N I
OH
Fig. 1. Structure
of the compounds
investigated.
200 -
plot for the oxidation Fig. 2. Lineweaver-Burk of the hydroxylamine (III) to the nitroxide (I); Km is 3.2 x 10m4 M; Vmax is 8.4 x 10-3 m-moles/min/~mole.
-I 50
2.5
-2.5
50 +,M-’
7.5
100
Fig. 3. Lineweaver-Burk plot for the inhibition of microsomal nitroxide reduction by various inhibitors: SKF-525A (0.018 mM) K. is 3.3 x 10W5 M. PCMA (0.25 mM) K! is 8.1 x 10m4 MI aniline (12.5 mM) K! is 1.9 x 10m2 M. PCMA is e-chloro-tj-methylaniline.
5.0
-2.5
2.5
50
,
z5
10.0
12.5
12.5
618
Preliminary Communications An alternative
alkyl oxidation uct.
this possiblity,
and identified
In conclusion, somal enzymes cytochrome vations
P-450 will reduce
cology
ring and subsequent the product
rearrangement
of the reduction
(I) via an enzyme
the nitroxide
with the findings
that basic amines
E. J. Rauckman
for the loss of the nitroxide
This research
(III)
is supported
different
is oxidized
than cytochrome
of Jenner cfl.(12)
prod-
by the microP-450 and that
(III).
These obser-
P-450-dependent
in part by a grant from N.I.H.
by a postdoctoral
is
by thin-layer
and Das and Ziegler
by a non-cytochrome
was supported
to a non-radical
was isolated
(I) to the hydroxylamine
are I-oxidized
signal
(III).
as the hydroxylamine
to the nitroxide
Acknowledgements-
account
we have shown that the hydroxylamine
are consistent
who suggest
which would
of the piperidine
To discount
chromatography
mechanism
National
Research
Service
(13) system.
No 10823.
Award
in Toxi-
No T32ES07002. REFERENCES
1.
H. S. Mason, A. -~ Rev. Biochem.
2.
3. R. Gillette,
3.
A. Stier and I. Reitz,
4.
A. Stier and I. Reitz, Naunyn-Schmiedebergs
5.
E. J. Rauckman,
6.
E. G. Rozantsev,
in Free Nitroxyl
7.
M. B. Abou-Donia
and J. W. Dieckert,
8.
0. H. Lowry,
9.
T. Omura and R. Sato, J_. -biol. Chem.
J. J. Kamm and H. A. Sesame, -___ Molec.
12. P. Jenner,
Xenobiotica
N. J. Rosebrough,
Arch. 9.
and C. H. Mitchell,
Radicals,
m.
Comn.
p. 93, Plenum
Toxic. appl.
239, 2379
Radicals, Archs
Archs -_
1958).
Pharmac.
p. 71, Plenum Biophys.
Xenobiotica
Biochem.
5, 409
270, R138
(1971).
1975).
Press, New York
(1970).
18, 507 (1971).
J. -biol. Chem. 193, 265 (1951).
(1964).
Biochem.
and A. H. Beckett,
13. M. L. Das and D. M. Ziegler,
4, 541
Path. Pharmak --
A. L. Farr and R. J. Randall,
in Free Nitroxyl
J. W. Gorrod
Pharmac.
1, 499 (1971).
G. M. Rosen and M. 6. Abou-Donia,
10. E. G. Rozantsev, 11. D. M. Ziegler
34, 595 (1965).
Biophys.
Press, New York
(1970).
150, 116 (1972).
3, 563 (1973). 140, 300 (1970).
Pharmacology,
Pergsmon
Vol. 26, pp. 675-678.
FORMATION
Printed
Press, 1977.
AND REDUCTION
in Great
OF A NITROXIDE
Britain
RADICAL
BY LIVER MICROSOMES
Gerald M. Rosen and Elmer J. Rauckman Department
of Physiology
and Pharmacology, Durham,
both a reducing non-specific reduction
P-450
is termed
enzyme
system
of molecular
act as a reducing substrate
replaces
Stable
(2).
low reactivity
Stier and Reitz
(3) have reported
reduced
in the presence
that (I) could methylpiperidine
(II)
stable equilibrium (e.p.r.)
spectroscopy,
reduction decided
to reinvestigate
The synthesis method
of Rauckman
chloroperbenzoic
__. et al
acid.
that an equilibrium
Reduction
For the drug induction
(III)
the
since they are paramagnetic,
and have a terminal
oxygen.
(I) (Fig. 1) is
They also reported oxidation
by electron
(4)
of 2,2,6,&tetra-
and oxyqen,
and that a
paramagnetic
the oxidation
resonance
of (II)
(3,4) gave no experimental
(I) was carried
and the
details,
out according
of 2,2,6,6_tetramethylpiperidine oxide and hydrogen
as reported solution
in the literature
(7).
we
to the
using E-
(II)
gave l-hydroxy-
The microsomes
such that a final concentration
The microsomal
of Lowry etz.(8)
protein
The liver microsomes
were isolated
(9)
were
of 0.5 g of
and cytochrome
and flmura and Sato
, rats were injected intraperitoneally
each day for 4 days.
to be reduced,
(6).
chloride
by the methods
P-450 is to
sequences.
was obtained.
studies
in which
enzymes
between
of (I) with platinum
were prepared
of suspension
tents were determined
(80 mg/kg)
of the microsomal
(5) by oxidation
in 1.15% potassium
liver/ml
by the NADPH-dependent
these reaction
other than the
if cytochrome
and NADPH.
of 2,2,6,6_tetramethylpiperidinoxyl
Liver microsomes
whole
oxygen
Since Stier and Reitz
2,2,6,6_tetramethylpiperidine
resuspended
conditions
of (I) could be observed
indicating
of (I) is achieved.
processes
that 2,2,6,6-tetramethylpiperidinoxyl
in the presence
concentration
in reducing
as oxygen analogs
under physiological
enzymatically
of
with the ferrohemochrome.
of liver microsomes,
be formed
the presence
nature of this relatively
intermediate,
can be considered
have relatively
Center,
1977)
since it requires
form of the cytochrome
in its reaction
radicals
31 January
The ambivalent
(1).
The reductive
is the ferrous
oxygen
nitroxide
oxygen
oxidase
has led to its implication
oxygen
agent,
1976; accepted
a mixed-function
agent and molecular
Medical
N.C. 2771n, U.S.A.
(Received 28 October
Cytochrome
Duke University
P-450 con-
respectively.
with phenobarbital as reported
earlier.
616 Electron
Preliminary Communications resonance spectra were obtained
Paramagnetic
E-9 spectrometer.
The kinetic
in the height of the central typical
(III),
experiment,
studies
were conducted
peak of the nitroxide
the reaction
medium
by measuring
triplet
contained
5 x 10
2.5 x 1o-4 M of NADPH, 0.1 ml of the microsomal
solution
contained
the final volume
using a Varian
-5
the decrease
as a function
M of the nitroxide
enzymes
These
experiments
were conducted
model
or increase
of time.
In a
or hydroxylamine
and sufficient
2.33 x 10 -2 M KH2P04 and 8.33 x low3 M MgC12 adjusted
to 0.5 ml.
Associates
buffer
(the
to pH 7.4) to bring
at ambient
temperature
in a
0.1 rnn flat cell. 2,2,6,6_Tetramethylpiperidine ambient
temperature
in the presence
used: the first containing except the microsomal the concentration Incubating oxygen
(1 x 1De4 M) was incubated
everything
enzymes.
solution
of the nitroxide
in the e.p.r.
oxygen was bubbled
(II)
of the amine
of 1 hr.
(II)
of the amine
to the nitroxide
with the microsomal
enzymes
These
observation
aliquots
experiments
suggested
(I) via (III)
of (II)
to (I).
in the presence
of air (lo), suggesting step in the conversion
nitroxide
formation
from the amine
aerobic
conditions
We feel that the enzyme
that cytochrome
oxidation
is not increased
inhibited
by SKF-525A
monoxide
min
by Ziegler
-1
by specific
responsible
and Mitchell
is not responsible
using phenobarbital-induced
led to the formation
measurable
pmole -' of cytochrome antagonists
amount of dithionite
e.p.r.
signal. min
-1
pmole
Evidence
for this oxidation: microsomes,
inhibitors
This reaction -1
for this oxidation (11).
of the
of is the
was gathered
(1) the rate of
(2) the oxidation
of cytochrome
undergoes
is not
P-450, and (3) carbon
by 50 per cent when added to dithionite-reduced
that the nitroxide
(III) at a kinetically
8.9 x 1o-3 m-moles
with a minimal
P-450
this reaction
We have also observed
competitively
described
as well as other specific
only inhibited
hydroxylamine
of its characteristic
of (III)
(II), incubation
to have a Km of 3.2 x 10 -4 M and V,,, of 8.4 x 1O-3 m-moles
to demonstrate
the
On the other hand, it is known
was found
amine oxidase
that either
(I).
by the appearance
mixed-function
through which
or that the rate of reduction
could be the rate-limiting
and NADPH under
(Fig. 2).
enzymes,
of an oxygen-saturated
from a mixture
as evidenced
P-450
and
(I) over that of the
nitroxide
cytochrome
were
spectrometer
of the microsomal
of the nitroxide
rapid autoxidation
we did not observe
at
everything
in an e.p.r.
in the presence
both by continuous
to the nitroxide
relatively
oxidation
Althouqh
(II)
than the rate of oxidation
(II)
were then placed
the concentration
a period
Two controls
and the second containing
cell for 30 min and by taking
during
undergoes
that enzymatic
NADPH,
enzymes
was determined.
was performed
is not oxidized
of (I) is greater that (III)
except
2,2,6,6_tetramethylpiperidine
This experiment
piperidine
(2.5 x 10e4 M) and oxygen.
These mixtures
and NADPH did not increase
control.
of NADPH
with the microsomal
an NADPH-dependent
reduction
microsomes. to the
rate with a Km of 1.8 x low4 M and a Vmax of P-450
of cytochrome
(Fig. 3). P-450.
and then exposing
This reduction
Pretreating
the enzymes
is inhibited
the microsomal
to carbon monoxide
enzymes prior
Preliminary
to introducing
the nitroxide
other hand, this reduction
and NADPH prevented
was greatly
were used in place of control
61-l
Communications
increased
the reduction
of the free radical.
if phenobarbital-induced
On the
microsomal
enzymes
enzymes.
-0 H
47
II
III
Y
N I
OH
Fig. 1. Structure
of the compounds
investigated.
200 -
plot for the oxidation Fig. 2. Lineweaver-Burk of the hydroxylamine (III) to the nitroxide (I); Km is 3.2 x 10m4 M; Vmax is 8.4 x 10-3 m-moles/min/~mole.
-I 50
2.5
-2.5
50 +,M-’
7.5
100
Fig. 3. Lineweaver-Burk plot for the inhibition of microsomal nitroxide reduction by various inhibitors: SKF-525A (0.018 mM) K. is 3.3 x 10W5 M. PCMA (0.25 mM) K! is 8.1 x 10m4 MI aniline (12.5 mM) K! is 1.9 x 10m2 M. PCMA is e-chloro-tj-methylaniline.
5.0
-2.5
2.5
50
,
z5
10.0
12.5
12.5
618
Preliminary Communications An alternative
alkyl oxidation uct.
this possiblity,
and identified
In conclusion, somal enzymes cytochrome vations
P-450 will reduce
cology
ring and subsequent the product
rearrangement
of the reduction
(I) via an enzyme
the nitroxide
with the findings
that basic amines
E. J. Rauckman
for the loss of the nitroxide
This research
(III)
is supported
different
is oxidized
than cytochrome
of Jenner cfl.(12)
prod-
by the microP-450 and that
(III).
These obser-
P-450-dependent
in part by a grant from N.I.H.
by a postdoctoral
is
by thin-layer
and Das and Ziegler
by a non-cytochrome
was supported
to a non-radical
was isolated
(I) to the hydroxylamine
are I-oxidized
signal
(III).
as the hydroxylamine
to the nitroxide
Acknowledgements-
account
we have shown that the hydroxylamine
are consistent
who suggest
which would
of the piperidine
To discount
chromatography
mechanism
National
Research
Service
(13) system.
No 10823.
Award
in Toxi-
No T32ES07002. REFERENCES
1.
H. S. Mason, A. -~ Rev. Biochem.
2.
3. R. Gillette,
3.
A. Stier and I. Reitz,
4.
A. Stier and I. Reitz, Naunyn-Schmiedebergs
5.
E. J. Rauckman,
6.
E. G. Rozantsev,
in Free Nitroxyl
7.
M. B. Abou-Donia
and J. W. Dieckert,
8.
0. H. Lowry,
9.
T. Omura and R. Sato, J_. -biol. Chem.
J. J. Kamm and H. A. Sesame, -___ Molec.
12. P. Jenner,
Xenobiotica
N. J. Rosebrough,
Arch. 9.
and C. H. Mitchell,
Radicals,
m.
Comn.
p. 93, Plenum
Toxic. appl.
239, 2379
Radicals, Archs
Archs -_
1958).
Pharmac.
p. 71, Plenum Biophys.
Xenobiotica
Biochem.
5, 409
270, R138
(1971).
1975).
Press, New York
(1970).
18, 507 (1971).
J. -biol. Chem. 193, 265 (1951).
(1964).
Biochem.
and A. H. Beckett,
13. M. L. Das and D. M. Ziegler,
4, 541
Path. Pharmak --
A. L. Farr and R. J. Randall,
in Free Nitroxyl
J. W. Gorrod
Pharmac.
1, 499 (1971).
G. M. Rosen and M. 6. Abou-Donia,
10. E. G. Rozantsev, 11. D. M. Ziegler
34, 595 (1965).
Biophys.
Press, New York
(1970).
150, 116 (1972).
3, 563 (1973). 140, 300 (1970).