Vol.
169,
May
31, 1990
No.
BIOCHEMICAL
1, 1990
AND
BIOPHYSICAL
RESEARCH
Pages
IN
ON THE DETECTION ADRIAMYCIN-PERFUSED
THE
OF PARAMAGNETIC RAT HEART:
B. Kalyanaramar?*
and
John
April
10,
30-38
SPECIES A REAPPRAISAL
E. Bakerb
Departments of ‘Radiology and bCardiothoracic Medical College of Wisconsin, 8701 Watertown Milwaukee, Wisconsin 53226 Received
COMMUNICATIONS
Surgery, Plank Road,
1990
SUMMARY. Recently, L. Costa et al. reported the direct detection of the superoxide anion and other paramagnetic species in the isolated, adriamycin-perfused rat heart [L. Costa et al. (1988) &&em. Biophys. Res. Commun. 153, 275-2801. We have reevaluated the results of their study and concluded that the ESR parameters of the spectrum obtained from the adriamycin-perfused heart are consistent with that of the peroxyl radical and not with that of the superoxide anion. In addition, the ESR spectrum of the peroxyl radical is very likely produced as an artifact caused by the grinding of myocardial tissue. This artifact may mask the ESR spectra of the adriamycin-derived semiquinone radical and the iron-sulfur protein components of myocardium. 0 1990 Academic
Press,
Inc.
The cardiotoxicity a one-electron
the above
sequence
a perferryl
species
ferritin
[15-171.
ferritin
in in vitro There
iron anion
has been attributed redox-cycling
peroxide
may
(HzOz)
of a more be present
to the formation
in the [l-11].
powerful
oxidant,
semiquinone
of a semiquinone,
of molecular
In the presence
as a contaminant
and the adriamycin
presence
either
of adventitious the hydroxyl
in the perfusate, have
oxygen
been shown
to iron,
radical
or released
to mobilize
or from
iron from
[15-171. interest
reperfusion shown
in the generation
[18-201.
Perfusion
of oxy-radicals of the isolated
in the rat
to generate
the hydroxyl
radical,
which
reported
the detection
of the superoxide
heart
isolated, with
redox
was subsequently
perfused active
heart
in the
quinones,
detected
such
by ESR-spin
[21].
perfused
et al. have
rat
hearts
in ESR
nitrogen
[22].
whom
Abbreviation
tubes
radical
correspondence nsed:
low-flow
ischemia
to the temperature
It is now
and other
recently
during
freeze-clamped
transferred
*To
and
hydrogen
Adventitious
systems
adriamycin undergoes
to the formation
the superoxide
has been
Costa
hearts,
and
leads
is a considerable
as adriamycin,
peroxyl
[12-141.
which
(0,:)
of reactions
of ischemia
trapping
anion
Both
drug
metabolite,
the superoxide
generate
context
of the antitumor
reduction
ESR;
to a liquid well
should Electron
of liquid
nitrogen
established
species
[22].
that
Th e investigators nitrogen,
Dewar,
and
mechanical
be addressed.
0006-291X/90 $1.50 Copyright 0 1990 by Academic Press, All rights of reproduction in any form
Resonance.
Inc. reserved.
the ESR grinding
[23-26).
Spin
were
30
anion
by direct
employed ground spectra of frozen
under
a processing liquid
recorded heart
ESR,
tissue
nitrogen
in adriamycin-
technique
in which
to a fine powder,
at the temperature
of liquid
can artifactually
generate
Vol.
169, No. 1, 1990
BIOCHEMICAL
Due to the enormous
interest
[27-301,
likely
organs
investigators. its impact
it
is very
Thus, upon
the results
for oxy
a consequence
of adriamycin
tissue
processirlg
masks
the appearance
associated metabolism
with
the
to determine
reported
In this communication base available
in the free radical-mediated that
it is critical
in their
we have
radicals,
metabolism
of adriamycin
in heart
study
their
NADH tissue
of drugs
by Costa
technique
and xenobiotics
in isolated
et al. may
be adopted
for tissue
processing
is valid,and
reported
by Costa
by
other
to assess
1221. the ESR parameters
whether
heart,
adriamycin
mitocbondrial
whether
RESEARCH COMMUNICATIONS
metabolism reported
the radical
in the ischemic
of the adriamycin-perfused
the
methodology
(i) compared
(ii) determined
of authentic
AND BIOPHYSICAL
heart and
(iii)
semiquinone
data
species
reported
by Costa
et al is generated
or is a peroxyl
radical,
generated
artifactually
whether
an artifactually
and the changes
dehydrogenase,
et al to the known
one of the enzymes
generated
accompanying proposed
as
during
peroxyl
radical
sulfur
protein
the iron
to be responsible
for
the
(31-331.
MATERIALS
AND
METHODS
Isolated Heart Model. Adult Sprague Dawley rats, maintained on a standard diet, were used for this study. Anesthesia was induced and maintained with Halothane (4% and l-2%, respectively), following which the right femoral vein was exposed and Heparin (150 IU/kg) a d ministered intravenously. After 1 min., the chest was opened via median sternotomy and the pericardium removed. The aorta and inferior vena cavae were isolated, and the heart was rapidly excised and placed in ice-cold perfusion medium. After 30 sec., the aorta was attached to a stainless steel cannula, the pulmonary artery wes incised to permit adequate coronary drainage, and the heart was perfused by the method of Langendorff 1341 a t a p er f usion pressure equivalent to 12kPa (90 mm Hg). A three-way tap, located immediately above the site of cannulation, allowed the entire perfusate to be diverted away from the heart to produce global, no-flow ischemia. Rectal temperature in the rat was found to be 37’ C. Normothermic perfusion w&s, therefore, carried out with perfusate at this temperature. The heart and perfusion fluids were kept in temperature-controlled chambers to maintain the myocardial temperature at the desired level. During all periods of ischemia, the heart was immersed in the perfusion medium used to infuse the coronary network. Perfusion Medium. The standard perfusion fluid was Krebs-Henseleit composition: 118.5 mM NaCl/25.0 mM NaHCOJ4.8 mM KCl/I.2 when gassed with 95% 0,/5% CO,) in which the calcium content 11.1 mM glucose. During the preparation of all calcium-containing the precipitation of calcium by gassing the solution with 5% CO, through cellulose acetate membranes of pore size, 5.0 pm.
bicarbonate buffer [35] having the following mM MgSO,. 7H,0/1.2 mM KHzPO, (pH 7.4 was reduced to 1.8 mM. To this was added solutions, precautions were taken to prevent Before use, all perfusion fluids were filtered
Tissue Preparation. Immediately after mounting on the cannula, hearts (n = 8 per group) were perfused aerobically for a 10 min. equilibration period. Hearts (ta = 8 per group) were then subjected to a 10 min. period of normothermic global ischemia. At the end of each perfusion period, hearts were freeze-clamped between stainless steel tongs (Biomedix, Elm Grove, WI) previously cooled to the temperature of liquid nitrogen. Frozen ventricular tissue was then processed for spectroscopic analysis by two separate methods. The first method of tissue processing involved grinding the frozen tissue to a fine powder using a stainless steel pestle and mortar (Biomedix) immersed in a liquid nitrogen bath. The fine powder was transferred under liquid nitrogen to a precision ESR tube (inside diameter, 3 mm; length, 30 mm) by placing the tube within the well of the mortar and dispensing the powdered tissue into the ESR tube with a spatula. The ESR tube was then immediately transferred to the lumen of a Dewar flask pre-cooled to the temperature of liquid nitrogen. The second method of tissue processing entailed chopping the wafer of frozen tissue under liquid nitrogen with a stainless steel spatula to produce small fragments (= 2 mm cubes). These fragments were immediately transferred to the lumen of a Dewar flask pre-cooled to the temperature of liquid nitrogen. Sample sizes with the two methods were approximately equal. All flasks containing the processed heart tissue were then placed in the resonance chamber of the spectrometer. Low-Temperature ESR metabolism of adriamycin
of Adriamycin catalyzed
Semiquinone. Semiquinones from adriamycin by xanthine and xanthine oxidase (Sigma Grade
31
were IV).
generated A typical
by anerobic incubation
Vol.
169,
No.
1, 1990
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
for ESR analysis consisted of adriamycin (100 @vi), xanthine (400 PM), and xanthine oxidase (0.1 unit) in 2 mL phosphate buffer (100 mM, pH 7.5 ). Incubations were prepared in duplicate. Immediate!y after detecting the ESR spectra of the adriamycin semiquinone or adriamycin aglycone at room temperature, frozen icicles (3-4 cm) of the same incubation mixture were prepared and supported in the lumen of a finger Dewar filled with liquid nitrogen.
ESR Spectroscopy. ESR spectra from all samples were recorded at the temperature of liquid nitrogen using a Varian E-109 spectrometer at 9.5 GHz and lOO-kHz field modulation. Magnetic field measurements were determined by using a Radiopan M.I-110 gaussmeter, and microwave frequency was measured by using an EiP 200 frequency counter. Hyperfine splitting was measured to 0.05 G directly from the magnetic field separations. All g value derivations were obtained from measurements of magnetic field and microwave frequency after correcting for the position of the gaussmeter probe.
RESULTS Direct
ESR
parameters
freeze-clamped
tissue
values factors
of the Superoxide from
hearts
= 2.03 and gJ. = 2.005). II such as complexation with
perfused
(g
of the superoxide
anion
(g]] = 2.03 and gL = 2.005)
reported
911-
values
Anion.
The metal under
with
adriamycin
g-value ions,
polarity
a variety
by Costa
et
- - Ba*+
O*’
-
-
ca*+
anion
of the medium, of experimental
TABLE OF TEE
[Fig.
lb in ref.221
to the superoxide is extremely
and temperature conditions.
al. do not correspond
g, value3
the ESR anion
sensitive [36-381.
data
H2O
2.065
0*:
Ethanol
2.08
0*;
H,O/CaCI,
2.05
OOH
H2O
2.035
that base
Hyperf!ne Coupling
were obtained
even in the presence
32
of Ca*+.
the
available
ANION
(G)
11.0
aAt least three different superoxide species were detected by ESR, depending upon the concentration of Ba*+. Although the structures of these species are not fully understood, is believed that there is some type of complexation between 02T and Ba*+. ESR spectra
Table
2.095 2.069 2.048
0,:
bSimiler
based
2.095 2.077 2.065
H20/DMSO/CaC12b
spectrum
of
on the g-
to environmental
It is evident
to the literature
I SUPEROXIDE
Medium
02:
report
was assigned
of the superoxide
ESR PARAMETERS Radical
In the previous
it
I lists
the
g-values for the
Vol.
169,
No. 1, 1990
superoxide
anion.
superoxide
anion.
Artifactual
Generation
Therefore,
can be generated this
effect
spectrum
assigned
ESR the
tissue
radical
(R,,
1).
these
not as a free radical
Parameters increase
intermediate
in oxy-radicals
radical
(g, = 2.04),
of mechanical
arising
from
Semiqninone
at Low
c. Ground
hearts
radical
verified
species.
In addition,
or chopped
(B) prior
be 77 K.
The ground
Spectrometer
of ischemic
to recording
conditions:
sample
rat heart
(gi,, = 2.002)
1 in ref.
microwave
power,
In their
during
low-flow
with
paper,
Costa
ischemia
200 G.
33
modulation
the frozen
of the sample
to 173 K (C) prior
2 mW;
a [23].
were
22) previously
the peroxyl
of the adriamycin-perfused
The temperature
was then warmed
ESR
heart
radical tissue,
of adriamycin.
freeze-clamped
of spectrum.
of ischemia,
possibly
et al. [22] attributed
to the redox-cycling
Sample
1. ESR spectrum
that
multi-line
. Samiquinone radical 0 Unknown ndiul 0 Pwoxyl radical A Iron SIMW cmtar A Secondary ublwmlquinone
Figure
species
and the ubisemiquinone (Fig.
radical
Temperatures.
in adriamycin-perfused
The
components
manipulation
the metabolism
radical
have
a semiquinone,
and an unidentified
the ESR spectral to the peroxyl
1).
not be the
radical
to 10 min.
(Fig.
components
that
others
subjected
different
of these
known
We and
heart
due to the peroxyl
that
be reassigned
as a consequence
of Adrlamycin
to consist
the absorptions
the rat
lb in ref. 22 could
It is well
[39,40].
are completely
analyzed
we conclude
should
from
for Fig.
Tissues.
grinding
ESR spectra
RESEARCH COMMUNICATIONS
responsible
Myoeardlal
or ground
a peroxyl
results,
anion
has been generated
The
been
only
species
by mechanical
chopped
= 2.004),
( cL 173 K),
From
In Ground
123-261.
previously
AND BIOPHYSICAL
the radical
tissues
tissue
has
to the superoxide
spectrum and
the frozen
temperatures (Fig.
tissue
that
Radical
in frozen
in heart
of ground
At higher evident
artifactually
with
ubisemiquinone
we conclude
of the Peroxyl
is present
freeze-clamped
BIOCHEMICAL
to recording
amplitude,
tissue
ground
was measured of spectrum.
2.0 G; scan range,
(A) to
of
Vol.
169,
No.
1, 1990
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
C”3
I Figure
2. The molecular
deoxyaglycone
semiquinones
derived
II
from
myocardial
tissue.
To
our
semiquinone
radicals
formed
structures
semiquinone
of adriamycin
(I) and adriamycin
(II).
ubiquinone
and
adriamycin.
knowledge,
the
existence
from
semiquinone
one-electron
However,
neither
of low-temperature
reduction
species (77
of adriamycin
K)
were ESR
detected spectra
has not been reported.
Room
I
?oom
Temperature
4
L, Ic
Liquid N2 -J-4
03
AHPP--+
Figure
3. ESR spectra
according
to experimental
conditions:
microwave
of adriamycin conditions power
Figure
4. ESR spectra
radical
II appeared,
with
1 mW;
of adriamycin time, from
semiquinone described
radical under
modulation deoxyaglcone
Materials
amplitude
b-
was generated
and Metbods.
Spectrometer
1 G; scan range 100 G.
semiquinone
the same incubation
34
I. This radical
mixture
II.
The ESR spectrum
in Fig. 3.
ground
derived
from
In order
10G
AHPP
in
of
to better
Vol.
169, No. 1, 1990
BIOCHEMICAL
AND BlOPHYSlCAL
RESEARCH COMMUNICATIONS
I
0
05 Figure
5. ReIationship
adriamycin Figure
semiquinone
the xanthine
spectra
/ xanthine
Adriamycin reductive
temperature). ESR
spectra The
system.
which
changed
Rapid
freezing
of I and
semiquinone to these
ESR
with ESR
asymmetrical
mixtures
(0).
intensity
nitrogen
temperature
we have
characterized
(I) of (A).
their
spectra
using
at room
saturated spectrum
ESR
(Fig.
environment of I (Fig.
spectrum
temperature
2) were
using
3, room
corresponding
in Figs.
generated
by the
the xanthine
temperature)
was
to II (Fig.
3 and 4 gave
4, room
the liquid
nitrogen
3 and 4. of I are 3.25 G (room
(AHJ
of I and 6).
II at
room
Comparison
of the endogenous
of the Iron-Sulfur the presence
(g = 2.02)
to the iron-sulfur NADH
one-line
and 6.5 G (77 K),
5 and
(I) of
temperature)
and 4.25
G (77 K),
and
AH,,
respectively. temperature
were
of the low
temperature
ubisemiquinone
radical
different
from
ESR
those
obtained
parameters
[23] reveal
considerable
The
spectrum
at liquid
of the adriamycin
differences
with
respect
parameters.
reveals
component
(Figs. that
Parameters
samples
curves
temperatures
to the
in Figs.
line widths
saturation
temperature
semiquinone(I1)
in a nitrogen
symmetrical
time
temperature)
tissues,
deoxyaglycone
temperature
of incubation
peak--to-peak
Power
at room
with
(0) and at liquid
intensity
system.
characteristic
II as shown
of II are 4.25 G (room
nitrogen
The
nitrogen
(P) in mW and the signal
in freeze-clamped
(I) and adriamycin
of adriamycin
oxidase
observed,
model
semiquinone
metabolism
/ xanthine first
oxidase
(P) in mW and the signal
power
temperature
species
pm
(A) and at liquid
the microwave
at room
of these
power
temperature
between
deoxyaglycone
the ESR
the microwave
at room
6. Relationship
adriamycin
understand
between
06
or succinate
in Myocardial
of a ubisemiquinone
(Fig.
center
Protein
1B). was
When
dehydrogenase
or a flavine
the scan range
observed.
Tissue.
These
semiquinone
was increased
iron-sulfur
center
ESR
(g = 2.0045)
of chopped and a reduced
to 1000 G, an additional peaks
are
associated
ischemic
heart
iron-sulfur
peak
(g = 1.94)
the
mitochondrial
with
due
[4i,42].
DISCUSSION This peroxyl
study
radical,
demonstrates not to that
that
the
of the superoxide
ESR
parameters anion
reported
as interpreted
35
by Costa in their
study.
et eI [22] correspond The peroxyl
radical
to that
of the
is generated
Vol.
BIOCHEMICAL
169, No. 1, 1990
artifactually
by mechanical
grinding
of frozen
during
the metabolism
of adriamycin
masks
the appearance
of the authentic
The oxygen
adriamycin
approximately rates
(i.e.,
tissue
6 ms [6]. 1 ml/min.)
using
The
ischemia is
also
in the
2.025
and
1.94 may
adriamycin. changes
respiratory
chain
in submitochondrial
It should, in the region
reduced
however,
because
tissue
adriamycin
to detect
this
radical
it may
heart
from
ischemic
mechanical
intensity heart
of the ESR
mitochondria
the
to no-flow
is catalyzed
center
of the
ESR
absorption
make
from
by
to iron-chelates
sulfur
freeze-clamped
will
signal
to detect
disproportionation-
iron
tissue
grinding
in heart
semiquinone
the dehydrogenase
by the reduced The
species
subjected
the
be
[6].
of bovine
of electrons
of
to
at low flow
of the adriamycin
because
At
estimated
be possible
and then
half-life
conditions
[31,32].
in chopped
with
adriamycin
peroxidation
of the overlap
radical
conditions.
perfused
with The
anaerobic been
its hydroquinone
via transfer
that
formed
peroxyl
has
However,
off).
to be catalyzed
be cautioned
g = 2.025
cut
and
particles
generated
species
[22].
perfused
lipid
has been shown
be considerably
reported
anaerobic
NADH-dependent
as an intermediate
totally
radical
in heart
is completely under
this
under
it is not feasible
tissue,
the adriamycin
metabolism
dehydrogenase
heart
supply
for
Therefore,
previously
not
the artifactually
by ESR
concentration
1 PM.
tissue,
radical.
half-life
between
that
Furthermore
detectable
considerably
equilibrium
dehydrogenase
NADH
the
oxygen than
oxygen
increased
Adriamycin
1 PM,
technique
the
It has been shown
(43).
greater
is only
in freeze-clamped
(when
coproportionation
NADH
of
intracellar
is far
semiquinone
radical
excess
heart.
RESEARCH COMMUNICATIONS
heart
adriamycin-semiquinone radical
the freeze-clamping
adriamycin global
in
adriamycin-perfused
in the ischemic
semiquinone
concentrations
AND BIOPHYSICAL
1 associated
after
it virtually
with
peaks
at g =
perfusion
with
impossible
to detect
the artifactually-generated
peroxyl
radical. Radical
species
during
mechanical
extent
of lipid
this,
may
grinding
detected
by Costa
perfused
hearts
strategies
the
the
processed
designed
increase
to investigate
I n vitro
[24].
ESR
spectral
microsomal
under
intensity
production
hypoxic
bond
of lipid
studies
have
hydroperoxides shown
work
was supported
that and
[17,44,45].
Based
on
in adriamycin-treated
heart
tissues,
as
of the artifactual
the experimental have
prior
rectified
technique their
to spectroscopic
free radical-mediated
by National
peroxyl
radical
spectral
analysis adriamycin
may
used
by Costa
in adriamycin-
et al. [22] is prone
misinterpretations. be of use in the
metabolism
The
use of frozen
development
in myocardium.
Institutes
of Health
Grants
HL-43030,
GM-29035
and
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the
conditions
ACKNOWLEDGMENT This
that
hearts.
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by chopping,
oxygen-oxygen
is the greatest
due to greater
reiterated
peroxyl
tissue
in the
to control
we have
of the unstable
by adriamycin
et al. is probably
as compared
generate
samples,
myocardial
induced that
In conclusion,
llrart
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R. G. (1989)
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Krebs,
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169,
May
31, 1990
No.
BIOCHEMICAL
1, 1990
AND
BIOPHYSICAL
RESEARCH
Pages
IN
ON THE DETECTION ADRIAMYCIN-PERFUSED
THE
OF PARAMAGNETIC RAT HEART:
B. Kalyanaramar?*
and
John
April
10,
30-38
SPECIES A REAPPRAISAL
E. Bakerb
Departments of ‘Radiology and bCardiothoracic Medical College of Wisconsin, 8701 Watertown Milwaukee, Wisconsin 53226 Received
COMMUNICATIONS
Surgery, Plank Road,
1990
SUMMARY. Recently, L. Costa et al. reported the direct detection of the superoxide anion and other paramagnetic species in the isolated, adriamycin-perfused rat heart [L. Costa et al. (1988) &&em. Biophys. Res. Commun. 153, 275-2801. We have reevaluated the results of their study and concluded that the ESR parameters of the spectrum obtained from the adriamycin-perfused heart are consistent with that of the peroxyl radical and not with that of the superoxide anion. In addition, the ESR spectrum of the peroxyl radical is very likely produced as an artifact caused by the grinding of myocardial tissue. This artifact may mask the ESR spectra of the adriamycin-derived semiquinone radical and the iron-sulfur protein components of myocardium. 0 1990 Academic
Press,
Inc.
The cardiotoxicity a one-electron
the above
sequence
a perferryl
species
ferritin
[15-171.
ferritin
in in vitro There
iron anion
has been attributed redox-cycling
peroxide
may
(HzOz)
of a more be present
to the formation
in the [l-11].
powerful
oxidant,
semiquinone
of a semiquinone,
of molecular
In the presence
as a contaminant
and the adriamycin
presence
either
of adventitious the hydroxyl
in the perfusate, have
oxygen
been shown
to iron,
radical
or released
to mobilize
or from
iron from
[15-171. interest
reperfusion shown
in the generation
[18-201.
Perfusion
of oxy-radicals of the isolated
in the rat
to generate
the hydroxyl
radical,
which
reported
the detection
of the superoxide
heart
isolated, with
redox
was subsequently
perfused active
heart
in the
quinones,
detected
such
by ESR-spin
[21].
perfused
et al. have
rat
hearts
in ESR
nitrogen
[22].
whom
Abbreviation
tubes
radical
correspondence nsed:
low-flow
ischemia
to the temperature
It is now
and other
recently
during
freeze-clamped
transferred
*To
and
hydrogen
Adventitious
systems
adriamycin undergoes
to the formation
the superoxide
has been
Costa
hearts,
and
leads
is a considerable
as adriamycin,
peroxyl
[12-141.
which
(0,:)
of reactions
of ischemia
trapping
anion
Both
drug
metabolite,
the superoxide
generate
context
of the antitumor
reduction
ESR;
to a liquid well
should Electron
of liquid
nitrogen
established
species
[22].
that
Th e investigators nitrogen,
Dewar,
and
mechanical
be addressed.
0006-291X/90 $1.50 Copyright 0 1990 by Academic Press, All rights of reproduction in any form
Resonance.
Inc. reserved.
the ESR grinding
[23-26).
Spin
were
30
anion
by direct
employed ground spectra of frozen
under
a processing liquid
recorded heart
ESR,
tissue
nitrogen
in adriamycin-
technique
in which
to a fine powder,
at the temperature
of liquid
can artifactually
generate
Vol.
169, No. 1, 1990
BIOCHEMICAL
Due to the enormous
interest
[27-301,
likely
organs
investigators. its impact
it
is very
Thus, upon
the results
for oxy
a consequence
of adriamycin
tissue
processirlg
masks
the appearance
associated metabolism
with
the
to determine
reported
In this communication base available
in the free radical-mediated that
it is critical
in their
we have
radicals,
metabolism
of adriamycin
in heart
study
their
NADH tissue
of drugs
by Costa
technique
and xenobiotics
in isolated
et al. may
be adopted
for tissue
processing
is valid,and
reported
by Costa
by
other
to assess
1221. the ESR parameters
whether
heart,
adriamycin
mitocbondrial
whether
RESEARCH COMMUNICATIONS
metabolism reported
the radical
in the ischemic
of the adriamycin-perfused
the
methodology
(i) compared
(ii) determined
of authentic
AND BIOPHYSICAL
heart and
(iii)
semiquinone
data
species
reported
by Costa
et al is generated
or is a peroxyl
radical,
generated
artifactually
whether
an artifactually
and the changes
dehydrogenase,
et al to the known
one of the enzymes
generated
accompanying proposed
as
during
peroxyl
radical
sulfur
protein
the iron
to be responsible
for
the
(31-331.
MATERIALS
AND
METHODS
Isolated Heart Model. Adult Sprague Dawley rats, maintained on a standard diet, were used for this study. Anesthesia was induced and maintained with Halothane (4% and l-2%, respectively), following which the right femoral vein was exposed and Heparin (150 IU/kg) a d ministered intravenously. After 1 min., the chest was opened via median sternotomy and the pericardium removed. The aorta and inferior vena cavae were isolated, and the heart was rapidly excised and placed in ice-cold perfusion medium. After 30 sec., the aorta was attached to a stainless steel cannula, the pulmonary artery wes incised to permit adequate coronary drainage, and the heart was perfused by the method of Langendorff 1341 a t a p er f usion pressure equivalent to 12kPa (90 mm Hg). A three-way tap, located immediately above the site of cannulation, allowed the entire perfusate to be diverted away from the heart to produce global, no-flow ischemia. Rectal temperature in the rat was found to be 37’ C. Normothermic perfusion w&s, therefore, carried out with perfusate at this temperature. The heart and perfusion fluids were kept in temperature-controlled chambers to maintain the myocardial temperature at the desired level. During all periods of ischemia, the heart was immersed in the perfusion medium used to infuse the coronary network. Perfusion Medium. The standard perfusion fluid was Krebs-Henseleit composition: 118.5 mM NaCl/25.0 mM NaHCOJ4.8 mM KCl/I.2 when gassed with 95% 0,/5% CO,) in which the calcium content 11.1 mM glucose. During the preparation of all calcium-containing the precipitation of calcium by gassing the solution with 5% CO, through cellulose acetate membranes of pore size, 5.0 pm.
bicarbonate buffer [35] having the following mM MgSO,. 7H,0/1.2 mM KHzPO, (pH 7.4 was reduced to 1.8 mM. To this was added solutions, precautions were taken to prevent Before use, all perfusion fluids were filtered
Tissue Preparation. Immediately after mounting on the cannula, hearts (n = 8 per group) were perfused aerobically for a 10 min. equilibration period. Hearts (ta = 8 per group) were then subjected to a 10 min. period of normothermic global ischemia. At the end of each perfusion period, hearts were freeze-clamped between stainless steel tongs (Biomedix, Elm Grove, WI) previously cooled to the temperature of liquid nitrogen. Frozen ventricular tissue was then processed for spectroscopic analysis by two separate methods. The first method of tissue processing involved grinding the frozen tissue to a fine powder using a stainless steel pestle and mortar (Biomedix) immersed in a liquid nitrogen bath. The fine powder was transferred under liquid nitrogen to a precision ESR tube (inside diameter, 3 mm; length, 30 mm) by placing the tube within the well of the mortar and dispensing the powdered tissue into the ESR tube with a spatula. The ESR tube was then immediately transferred to the lumen of a Dewar flask pre-cooled to the temperature of liquid nitrogen. The second method of tissue processing entailed chopping the wafer of frozen tissue under liquid nitrogen with a stainless steel spatula to produce small fragments (= 2 mm cubes). These fragments were immediately transferred to the lumen of a Dewar flask pre-cooled to the temperature of liquid nitrogen. Sample sizes with the two methods were approximately equal. All flasks containing the processed heart tissue were then placed in the resonance chamber of the spectrometer. Low-Temperature ESR metabolism of adriamycin
of Adriamycin catalyzed
Semiquinone. Semiquinones from adriamycin by xanthine and xanthine oxidase (Sigma Grade
31
were IV).
generated A typical
by anerobic incubation
Vol.
169,
No.
1, 1990
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
for ESR analysis consisted of adriamycin (100 @vi), xanthine (400 PM), and xanthine oxidase (0.1 unit) in 2 mL phosphate buffer (100 mM, pH 7.5 ). Incubations were prepared in duplicate. Immediate!y after detecting the ESR spectra of the adriamycin semiquinone or adriamycin aglycone at room temperature, frozen icicles (3-4 cm) of the same incubation mixture were prepared and supported in the lumen of a finger Dewar filled with liquid nitrogen.
ESR Spectroscopy. ESR spectra from all samples were recorded at the temperature of liquid nitrogen using a Varian E-109 spectrometer at 9.5 GHz and lOO-kHz field modulation. Magnetic field measurements were determined by using a Radiopan M.I-110 gaussmeter, and microwave frequency was measured by using an EiP 200 frequency counter. Hyperfine splitting was measured to 0.05 G directly from the magnetic field separations. All g value derivations were obtained from measurements of magnetic field and microwave frequency after correcting for the position of the gaussmeter probe.
RESULTS Direct
ESR
parameters
freeze-clamped
tissue
values factors
of the Superoxide from
hearts
= 2.03 and gJ. = 2.005). II such as complexation with
perfused
(g
of the superoxide
anion
(g]] = 2.03 and gL = 2.005)
reported
911-
values
Anion.
The metal under
with
adriamycin
g-value ions,
polarity
a variety
by Costa
et
- - Ba*+
O*’
-
-
ca*+
anion
of the medium, of experimental
TABLE OF TEE
[Fig.
lb in ref.221
to the superoxide is extremely
and temperature conditions.
al. do not correspond
g, value3
the ESR anion
sensitive [36-381.
data
H2O
2.065
0*:
Ethanol
2.08
0*;
H,O/CaCI,
2.05
OOH
H2O
2.035
that base
Hyperf!ne Coupling
were obtained
even in the presence
32
of Ca*+.
the
available
ANION
(G)
11.0
aAt least three different superoxide species were detected by ESR, depending upon the concentration of Ba*+. Although the structures of these species are not fully understood, is believed that there is some type of complexation between 02T and Ba*+. ESR spectra
Table
2.095 2.069 2.048
0,:
bSimiler
based
2.095 2.077 2.065
H20/DMSO/CaC12b
spectrum
of
on the g-
to environmental
It is evident
to the literature
I SUPEROXIDE
Medium
02:
report
was assigned
of the superoxide
ESR PARAMETERS Radical
In the previous
it
I lists
the
g-values for the
Vol.
169,
No. 1, 1990
superoxide
anion.
superoxide
anion.
Artifactual
Generation
Therefore,
can be generated this
effect
spectrum
assigned
ESR the
tissue
radical
(R,,
1).
these
not as a free radical
Parameters increase
intermediate
in oxy-radicals
radical
(g, = 2.04),
of mechanical
arising
from
Semiqninone
at Low
c. Ground
hearts
radical
verified
species.
In addition,
or chopped
(B) prior
be 77 K.
The ground
Spectrometer
of ischemic
to recording
conditions:
sample
rat heart
(gi,, = 2.002)
1 in ref.
microwave
power,
In their
during
low-flow
with
paper,
Costa
ischemia
200 G.
33
modulation
the frozen
of the sample
to 173 K (C) prior
2 mW;
a [23].
were
22) previously
the peroxyl
of the adriamycin-perfused
The temperature
was then warmed
ESR
heart
radical tissue,
of adriamycin.
freeze-clamped
of spectrum.
of ischemia,
possibly
et al. [22] attributed
to the redox-cycling
Sample
1. ESR spectrum
that
multi-line
. Samiquinone radical 0 Unknown ndiul 0 Pwoxyl radical A Iron SIMW cmtar A Secondary ublwmlquinone
Figure
species
and the ubisemiquinone (Fig.
radical
Temperatures.
in adriamycin-perfused
The
components
manipulation
the metabolism
radical
have
a semiquinone,
and an unidentified
the ESR spectral to the peroxyl
1).
not be the
radical
to 10 min.
(Fig.
components
that
others
subjected
different
of these
known
We and
heart
due to the peroxyl
that
be reassigned
as a consequence
of Adrlamycin
to consist
the absorptions
the rat
lb in ref. 22 could
It is well
[39,40].
are completely
analyzed
we conclude
should
from
for Fig.
Tissues.
grinding
ESR spectra
RESEARCH COMMUNICATIONS
responsible
Myoeardlal
or ground
a peroxyl
results,
anion
has been generated
The
been
only
species
by mechanical
chopped
= 2.004),
( cL 173 K),
From
In Ground
123-261.
previously
AND BIOPHYSICAL
the radical
tissues
tissue
has
to the superoxide
spectrum and
the frozen
temperatures (Fig.
tissue
that
Radical
in frozen
in heart
of ground
At higher evident
artifactually
with
ubisemiquinone
we conclude
of the Peroxyl
is present
freeze-clamped
BIOCHEMICAL
to recording
amplitude,
tissue
ground
was measured of spectrum.
2.0 G; scan range,
(A) to
of
Vol.
169,
No.
1, 1990
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
C”3
I Figure
2. The molecular
deoxyaglycone
semiquinones
derived
II
from
myocardial
tissue.
To
our
semiquinone
radicals
formed
structures
semiquinone
of adriamycin
(I) and adriamycin
(II).
ubiquinone
and
adriamycin.
knowledge,
the
existence
from
semiquinone
one-electron
However,
neither
of low-temperature
reduction
species (77
of adriamycin
K)
were ESR
detected spectra
has not been reported.
Room
I
?oom
Temperature
4
L, Ic
Liquid N2 -J-4
03
AHPP--+
Figure
3. ESR spectra
according
to experimental
conditions:
microwave
of adriamycin conditions power
Figure
4. ESR spectra
radical
II appeared,
with
1 mW;
of adriamycin time, from
semiquinone described
radical under
modulation deoxyaglcone
Materials
amplitude
b-
was generated
and Metbods.
Spectrometer
1 G; scan range 100 G.
semiquinone
the same incubation
34
I. This radical
mixture
II.
The ESR spectrum
in Fig. 3.
ground
derived
from
In order
10G
AHPP
in
of
to better
Vol.
169, No. 1, 1990
BIOCHEMICAL
AND BlOPHYSlCAL
RESEARCH COMMUNICATIONS
I
0
05 Figure
5. ReIationship
adriamycin Figure
semiquinone
the xanthine
spectra
/ xanthine
Adriamycin reductive
temperature). ESR
spectra The
system.
which
changed
Rapid
freezing
of I and
semiquinone to these
ESR
with ESR
asymmetrical
mixtures
(0).
intensity
nitrogen
temperature
we have
characterized
(I) of (A).
their
spectra
using
at room
saturated spectrum
ESR
(Fig.
environment of I (Fig.
spectrum
temperature
2) were
using
3, room
corresponding
in Figs.
generated
by the
the xanthine
temperature)
was
to II (Fig.
3 and 4 gave
4, room
the liquid
nitrogen
3 and 4. of I are 3.25 G (room
(AHJ
of I and 6).
II at
room
Comparison
of the endogenous
of the Iron-Sulfur the presence
(g = 2.02)
to the iron-sulfur NADH
one-line
and 6.5 G (77 K),
5 and
(I) of
temperature)
and 4.25
G (77 K),
and
AH,,
respectively. temperature
were
of the low
temperature
ubisemiquinone
radical
different
from
ESR
those
obtained
parameters
[23] reveal
considerable
The
spectrum
at liquid
of the adriamycin
differences
with
respect
parameters.
reveals
component
(Figs. that
Parameters
samples
curves
temperatures
to the
in Figs.
line widths
saturation
temperature
semiquinone(I1)
in a nitrogen
symmetrical
time
temperature)
tissues,
deoxyaglycone
temperature
of incubation
peak--to-peak
Power
at room
with
(0) and at liquid
intensity
system.
characteristic
II as shown
of II are 4.25 G (room
nitrogen
The
nitrogen
(P) in mW and the signal
in freeze-clamped
(I) and adriamycin
of adriamycin
oxidase
observed,
model
semiquinone
metabolism
/ xanthine first
oxidase
(P) in mW and the signal
power
temperature
species
pm
(A) and at liquid
the microwave
at room
of these
power
temperature
between
deoxyaglycone
the ESR
the microwave
at room
6. Relationship
adriamycin
understand
between
06
or succinate
in Myocardial
of a ubisemiquinone
(Fig.
center
Protein
1B). was
When
dehydrogenase
or a flavine
the scan range
observed.
Tissue.
These
semiquinone
was increased
iron-sulfur
center
ESR
(g = 2.0045)
of chopped and a reduced
to 1000 G, an additional peaks
are
associated
ischemic
heart
iron-sulfur
peak
(g = 1.94)
the
mitochondrial
with
due
[4i,42].
DISCUSSION This peroxyl
study
radical,
demonstrates not to that
that
the
of the superoxide
ESR
parameters anion
reported
as interpreted
35
by Costa in their
study.
et eI [22] correspond The peroxyl
radical
to that
of the
is generated
Vol.
BIOCHEMICAL
169, No. 1, 1990
artifactually
by mechanical
grinding
of frozen
during
the metabolism
of adriamycin
masks
the appearance
of the authentic
The oxygen
adriamycin
approximately rates
(i.e.,
tissue
6 ms [6]. 1 ml/min.)
using
The
ischemia is
also
in the
2.025
and
1.94 may
adriamycin. changes
respiratory
chain
in submitochondrial
It should, in the region
reduced
however,
because
tissue
adriamycin
to detect
this
radical
it may
heart
from
ischemic
mechanical
intensity heart
of the ESR
mitochondria
the
to no-flow
is catalyzed
center
of the
ESR
absorption
make
from
by
to iron-chelates
sulfur
freeze-clamped
will
signal
to detect
disproportionation-
iron
tissue
grinding
in heart
semiquinone
the dehydrogenase
by the reduced The
species
subjected
the
be
[6].
of bovine
of electrons
of
to
at low flow
of the adriamycin
because
At
estimated
be possible
and then
half-life
conditions
[31,32].
in chopped
with
adriamycin
peroxidation
of the overlap
radical
conditions.
perfused
with The
anaerobic been
its hydroquinone
via transfer
that
formed
peroxyl
has
However,
off).
to be catalyzed
be cautioned
g = 2.025
cut
and
particles
generated
species
[22].
perfused
lipid
has been shown
be considerably
reported
anaerobic
NADH-dependent
as an intermediate
totally
radical
in heart
is completely under
this
under
it is not feasible
tissue,
the adriamycin
metabolism
dehydrogenase
heart
supply
for
Therefore,
previously
not
the artifactually
by ESR
concentration
1 PM.
tissue,
radical.
half-life
between
that
Furthermore
detectable
considerably
equilibrium
dehydrogenase
NADH
the
oxygen than
oxygen
increased
Adriamycin
1 PM,
technique
the
It has been shown
(43).
greater
is only
in freeze-clamped
(when
coproportionation
NADH
of
intracellar
is far
semiquinone
radical
excess
heart.
RESEARCH COMMUNICATIONS
heart
adriamycin-semiquinone radical
the freeze-clamping
adriamycin global
in
adriamycin-perfused
in the ischemic
semiquinone
concentrations
AND BIOPHYSICAL
1 associated
after
it virtually
with
peaks
at g =
perfusion
with
impossible
to detect
the artifactually-generated
peroxyl
radical. Radical
species
during
mechanical
extent
of lipid
this,
may
grinding
detected
by Costa
perfused
hearts
strategies
the
the
processed
designed
increase
to investigate
I n vitro
[24].
ESR
spectral
microsomal
under
intensity
production
hypoxic
bond
of lipid
studies
have
hydroperoxides shown
work
was supported
that and
[17,44,45].
Based
on
in adriamycin-treated
heart
tissues,
as
of the artifactual
the experimental have
prior
rectified
technique their
to spectroscopic
free radical-mediated
by National
peroxyl
radical
spectral
analysis adriamycin
may
used
by Costa
in adriamycin-
et al. [22] is prone
misinterpretations. be of use in the
metabolism
The
use of frozen
development
in myocardium.
Institutes
of Health
Grants
HL-43030,
GM-29035
and
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