Vol.
179,
No.
September
30,
3, 1991
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
1991
COMMUNICATIONS Pages
1305-1310
LIGHT-DEPENDENT SPIN TRAPPING OF HYDROXYL RADICAL FROM HUMAN ERYlHROCYTES Leon A. Bynoel, Sovitj Pous, John D. Gottschl,
and Gerald M. Rosena’
’ Wilmer Institute, Johns Hopkins Hospital, 600 N. Wolfe Street, Baltimore, Md. 21205 2 University of Maryland School of Pharmacy, 20 N. Pine Street, Baltimore, Md. 21201 Received
August
9, 1991
Summary:
The generation of reactive oxygen species from human erythrocytes has previously been demonstrated. Furthermore, erythrocytic protoporphyrin IX has been shown to generate superoxide and singlet oxygen when exposed to light. These findings suggest that a component of erythrocytic reactive oxygen species By inhibiting erythrocyte superoxide production may be light-dependent. dismutase, catalase, and glutathione peroxidase with N,N-diethyldithiocarbamate or sodium cyanide, we demonstrate the light-dependent generation of hydroxyl radical in human erythrocytes using spin trapping/Electron Spin Resonance spectroscopy. This finding may be significant in tissues where blood is exposed 0 1991 Academic Press,Inc. to light, such as in the eye.
Age related macular degeneration and the retinopathy of prematurity are disordersof the retina in which light exposure has been implicated as a risk factor (1,2). For this reason, a common etiology for these diseaseshas been proposed (3). According to this hypothesis, light exposure of erythrocytes in blood vessels supplyingthe retina activates photoactive compounds, such as protoporphyrin IX (PP IX), the precursormoleculeof heme and a normal componentof erythrocytes (4). Transfer of an electron and energy to oxygen results in the formation of superoxide (‘02) and singlet oxygen (‘02) respectively (3,5). In a recent study (3), we demonstrated the wavelength-dependent photogeneration of 02-’ and 102 by PPIX at physiological concentrations using spin trapping techniques/Electron Spin Resonance (ESR) spectroscopy (6-8). Based on these findings, we propose that a similar process might occur in erythrocytes. However, unlike our earlier studies with homogenous solutions, erythrocytes contain numerousenzymes capableof metabolizingthe spin trap and/or the spin trapped adduct, Therefore, a new spin trapping system has been developed,which is considerablemore sensitive and more stable than other spin trapping proceduresto study the formation of hydroxyl radical
’ To whom
correspondence
Abbrevratrons : PP diethylenetriaminepentaacetic isothiocyano-2,2’-disulfonic N-tert-butylnitrone; DMPO,
should
be addressed.
IX,
protoporphyrin IX; ESR, electron acid; DETC, N,N-diethyldithiocarbamate; acid stilbene; SOD, superoxide dismutase; 5,5-dimethyl-1-pyrroline-N-oxide.
1305
Copyright Ail rights of
spin
resonance; DTPA, SITS, 4-acetamido-4’POBN, a-(4-pyridyl-1 -oxide)-
0006-291X/91 $1.50 0 1991 by Academic Press, Inc. reproduction
in any
form
reserved.
Vol.
BIOCHEMICAL
179, No. 3, 1991
(OH.)
in metabolically
new spin trapping
method to study the photogeneration
RESEARCH COMMUNICATIONS
In this communication,
active cells such as erythrocytes.
so doing, we are able to determine radical
AND BIOPHYSICAL
of OH in light-exposed
the compartmentalization
we apply this erythrocytes.
In
of the spin trap and the site of free
formation.
MATERIALS AND METHODS
Reagents Diethylenetriaminepentaacetic acid (DTPA), sodium cyanide (NaCN), N,N-diethyldithiocarbamate (DETC), 4-acetamido-4’-isothiocyano-2,2’-disulfonic acid stil-bene (SITS) and catalase were purchased from Sigma Chemical Co. (St. Louis, MO). Superoxide dismutase (SOD) was purchased from Boehringer Mannheim (Indianapolis, IN). The spin trap a-(4-pyridyl-I -oxide)-N-tert-butylnitrone (POBN) was purchased from Aldrich Chemical Co. (Milwaukee. WI). The spin trap 5,5-dimethyl-l-pyrroline-N-oxide (DMPO) and 5cyano-l-hydroxyl-2,2-dimethylpyrroli-dine were synthesized according to the method of Bonnett, et a/ (9). Air oxidation of the cyan0 pyrrolidine gave 5cyano-2,2-dimethyl-lpyrrolidinyloxy (DMPO-CN). All buffers were passed through a Chelex-100 ion exchange column (Eiorad, Richmond, CA) to remove trace metal ion impurities (10).
Protoporphyrin Determination Erythrocyte and plasma protoporphyrin the method of Chisolm, Jr. and Brown (11).
IX (PP IX) levels were determined
according
to
Detection of hydroxyl radical In a darkened room, blood was collected from four volunteers and heparin was added to each blood sample at 25 U/ml. The blood was then centrifuged (400 x g for 5 minutes at 40 C) and plasma and leukocytes were removed. The pelleted erythrocytes were washed three times in isotonic saline. Following the final centrifugation, a portion of the washed erythrocytes were resuspended I:1 in phosphate-buffered saline pH = 7.4 (PBS) containing 1 g/L glucose (PBSG). The remaining erythrocytes, which were used for spin trapping experiments, were resuspended 1:l in PBSG with 2 mM DTPA (PBSG-DTPA) (12). In separate trials, resuspended erythrocytes were incubated with either: 1) 50 mM DETC (inhibits SOD, catalase, and glutathione peroxidase; (13)) for 10 minutes at 25O C, followed by three washes and resuspension in PBSG-DTPA; or 2) 50 mM DETC followed by three washes and incubation at 37O C with 60 uM SITS (anion channel blocker; (14)) for 2 hours. After SITS treatment, erythrocytes were washed three times and resuspended in PBSG-DTPA. A control group of erythrocytes were incubated in PBSG. Following the incubations, one of the two spin traps (200 mM DMPO or 20 mM POBN with 1% ethanol) was added to the resuspended erythrocytes. In several experiments where DMPO was used, NaCN was added to the reaction mixture at either 50 mM or 100 mM just prior to the addition of DMPO. In all experiments, the final volume of the reaction mixture was 500 ul. The reaction mixtures described above were then transferred to a flat quartz ESR cell, fitted into the cavity of an ESR spectrometer (Varian Associates model E-9, Palo Alto, CA) and spectra were recorded at 20° C in the dark. After this, approxi-mately 2000 pW/cm2 of continuous irradiation from a 150 watt light source was delivered to the ESR cavity and spectra were again recorded at set time intervals.
RESULTS AND DISCUSSION In our earlier species
paper, we demonstrated
the light-dependent
from PP IX (3). Based on these findings
production
of reactive
oxygen
and the fact that human
erythrocytes
contain
this porphyrin (in this study, the erythrocytic PP IX levels of the four subjects were in the normal range, 35.3 L 3 ugldl; (5)), we determined the nature of the free radicals generated during
exposure of erythrocytes to light. Previous studies with erythrocytes
cytochrome
c reduction
method
(15,16).
have
measured
However, 1306
when
the production erythrocytes
of ‘0~~ were
using the
incubated
with
Vol.
179, No. 3, 1991
Figure DMPO.
BIOCHEMICAL
ESR spectra
resulted
from
AND BIOPHYSICAL
the irradiation
of erythrocytes
RESEARCH COMMUNICATIONS
in the presence
of NaCN
and
Scan A was obtained after 8 minutes of irradiation of erythrocytes in PBSG-DTPA with 50 mM NaCN and 200 mM DMPO. Scan B was obtained under the same conditions as A. except the reaction mixture was not exposed to light. Scan C was obtained under the same conditions as A, except the concentration of NaCN was 100 mM. If the reaction mixture did not contain NaCN, no spectrum was observed, even with light exposure duration of 2 hours. Light intensity was 2000 uW/cm2. Microwave power was 20 mW, modulation frequency was 100 kHz with an amplitude of 1 .O G, sweep time was 12.5 G/min and the receiver gain was 2 x 1 Cl4with a response time of 1 sec. Scan D is a computer simulation of the ESR spectrum of a 2.5:1 mixture of DMPO-OH and DMPO-CN. Scan E is a.computer simulation of the ESR spectrum of a 1.3:1 mixture of DMPO-DH and DMPO-CN. The hyperfine splitting constants for DMPO-OH are AN = AH = 14.9 G; for DMPO-CN, AN = 15.4 G and AH = 18.9 G. DMPO, was
we were unable
not surprising,
detecting
to spin trap ‘0~~, even in the presence
considering
this free radical
approximately
between
(e.g. at pH = 7.4, the rate of ‘02‘
reduction
IO5 M-l set -l; the reaction
set -I, while
reactions
self-dismutation
order to detect any free radicals use inhibitors peroxide.
of light (data not shown).
rate constants
well as the rate of competing M-l
the disparate
of the enzymes
One such compound
of ‘0~~ with DMPO
regulate
is NaCN.
With DMPO 1307
c is
by SOD is 2 x log
Therefore,
using spin trapping
intracellular
of
is only 10 M-t set -l; (17)) as
set - t ; (18.19)).
from erythrocytes
which
of cytochrome
for ‘0~~ (e.g. the rate of dismutation
is lo5 M-l
This
the two methods
we felt that in
techniques,
levels of superoxide in the reaction
mixture,
we had to
and hydrogen light-exposed
Vol.
BIOCHEMICAL
179, No. 3, 1991
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
, 1OG ,
EiQure 7 ESR spectrum of DMPO-CN. The reaction mixture contained DMPO-CN in PBS (pH=7.8) with 1 mM DTPA. ESR settings were identical to those in figure 1, except the sweep time was 25 G/min and the receiver gain was 1.25 x 10 3. DMPO-CN was synthesized by the copper acetate/On oxidation of 5-cyano-1 -hydroxyl-2,2-dimethylpyrrolidine according to the method of Bonnett, et al (9). erythrocytes trapped
treated
adducts,
spectrum
with NaCN produced
DMPO-OH
was observed
simulations
an ESR spectrum
and DMPO-CN
(1B).
Verfication
are a number
of possible
the presence
synthesis
sources of DMPO-OH
assignment of DMPO-CN
OH and DMPO-CN Even
though
DMPO/EtOH
(Pou,
peroxide
on computer
(fig. 2) (9). While there
for both DMPO-OH
of NaCN, ‘OH, but not ‘OS-, has been shown to generate system, hydrogen
of light no ESR
is based
(for a review of this subject see Pou, et a/. (8)),
case ‘OH is responsible
which reacts with DMPO to give DMPO-CN. generating
of a mixture of two spin
(fig. IA and C). In the absence of the above
(20) (1D and E) and independent
we believe that in this particular
composed
Also, the addition
(Hs02)
and DMPO-CN.
In
cyano free radical (‘CN),
of DMPO and NaCN to the ‘OH-
and ferrous salts, produces
a mixture of DMPO-
(fig. 3). POBN/EtOH
is IO times
et al, unpublished
more
sensitive
for spin
trapping
data), we again had to use an inhibitor
‘OH
than
of erythrocytic
antioxidant enzymes in order to spin trap ‘OH. Erythrocytes pretreated with DETC produced the characteristic POBN-CH(CH)sOH adduct spectrum only when the mixture was exposed to light (fig. 4A). The addition However, SITS (14)
if DETC-treated no ESR spectrum
Because produced
erythrocytes
to the reaction mixture did not change
had also been treated
was observed
is at least partially dependent
will be dismutated
(either
the spectrum.
with the anion channel
blocker
(fig. 48).
the detection of ‘OH requires the inhibition
that ‘OH production ‘02-
of SOD and catalase
of erythrocyte
on the generation
spontaneously
or by SOD)
SOD, these data suggest of ‘02-. In the erythrocyte, to H202
(18,lS)
which,
Figure ESR spectrum resulted from the addition of NaCN to hydrogen peroxide and iron. The reaction mixture contained 0.3% HaOa, 0.1 mM Fe 2+, and 50 mM NaCN in PBSG-DTPA. ESR settings were identical to those in figure 1. 1308
Vol.
179, No. 3, 1991
BIOCHEMICAL
AND BIOPHYSICAL
A
11
4I
RESEARCH COMMUNICATIONS
B)
from fhe irradiation of DETC-treated etyythrocytes in the Scan A was obtained after a 20 minute irradiation of DETCpretreated erythrocytes suspended in PBSG-DTPA with 20 mM POBN and 1% EtOH. The addition of SOD (100 U/ml) and catalase (100 U/ml) had no effect on the scan. Light intensity and ESR settings were as described in Figure 1. Scan B was obtained under the same conditions as A, except erythrocytes were also pretreated with the anion channel blocker SITS. Scan C is a computer simulation of the ESR spectrum for POBN-EtOH. The hyperfine splitting constants for POBN-EtOH are AN = 15.6 G and AH = 2.7 G. Figure presence
ESR spectra resuited of POBN and EtOH.
&ure 5, ESR spectra resulted from the irradiation of the supernarant erythrocytes in the presence of POBN and EfOH. Erythrocytes were treated,
of
DETC-treated
suspended in POBNEtOH, and exposed to light as described in figure 4A. Following irradiation, the reaction mixture was centrifuged at 400 x g and the supernatant was scanned with ESR, producing scan A. No additional irradiation of the supernatant fraction was required to produce this scan. The erythrocyte fraction produced a similar scan. When there was no irradiation of the reaction mixture prior to isolation of the supernatant, a 30 minute light exposure of the supernatant produced scan B. Light intensity and ESR settings were as described in Figure 1. along with ‘0~~ (in the presence normal
antioxidant
enzymes,
of ferric (Fe3+) salts will generate
Hz02 is reduced
of SOD would slow the rate of conversion allow
the accumula-tion
peroxidase
(13).
generation
These
Our
data
preventing
support
that
effect
by either
were performed.
EtOH and then centrifuged, supernatant,
resulted
mixture
fractions,
results
membrane.
suggest
exhibited that both
Exactly
of iron
the extracellular
inside
the
erythrocyte
is
of ‘OH. SITS may
erythrocytes of the cellular
were
from
trapped
with POBN and
and not of the remaining
into cellular
we suggest that light-dependent
or
additional
However,
if the
and supernatant
of POBN-CH(CH)30H
and POBN-CH(CH)sOH
and a portion
occurs,
(data not shown).
characteristic
spin
erythrocytes
incubated
fraction,
to light and then separated
the erythrocyte,
favor the
the detection
which of the above processes
ESR spectra
would
and glutathione
erythrocytes,
flux of ‘OH
of ‘OH by POBN/EtOH
POBN
of erythrocytes
catalase
by spin trapping.
within
When DETC-treated
was first exposed
inhibits
with SITS inhibits
only light exposure
Based on these findings,
extracellular
(fig. 5).
traverse
the erythro-cytic
formation
and spin trapping
of the spin trapped
adduct diffuses into the
milieu. what
not appear
erythrocyte
blocking
in spin trapping
both fractions
of ‘OH occurs within
does
produced
blockade
the influx of POBN. To determine
experiments
These
also partially
and the presence
‘OH
since anion channel
this
reaction
of ‘02- to H202, DETC-treatment
since DETC
conditions
with
to water (21). While it is true that the inhibition
of ‘OH to a level which allows its detection
intracellularly, cause
of Hz02
‘OH. In erythrocytes
role, if any, photogenerated to escape
itself, perhaps
intact
‘OH plays in tissue injury
erythrocytes.
by causing
However,
the peroxidation 1309
is unclear
‘OH may mediate of membrane
lipids
damage
since it to the
(22). This might
Vol.
179,
No.
3, 1991
result in hemolysis generate
and increased
free radicals
erythrocytic disorders
‘OH
BIOCHEMICAL
AND
BIOPHYSICAL
escape of photoactive
compounds
(i.e. ‘02’ and ‘OH) extracellularly.
may contribute
to tissue
injury
RESEARCH
COMMUNICATIONS
(i.e. PP IX) which can then
By this mechanism,
and therefore
photogenerated
play an important
role in
of the retina.
ACKNOWLEDGMENTS The authors wish to thank Erik V. Rents and Dr. Gary R. Buettner for their help with the computer simulations and Veronica Kestenberg for performing the protoporphyrin IX assays. This research was supported by grants from the National Institute of Health (HL33550) and Tissue Banks International.
REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9.
Munoz, B., West, S., Bressler, N., Bressler, S., Rosenthal, F.S., and Taylor, H.R. (1990) Invest. Ophthalmol. Vis. Sci. (abstracts) 31(4), 49. Glass, P., Avery, G.B., Subramanian, K.N., Keys, M.P., Sostek, A.M., and Friendly, D.S. (1985) N. Engl. J. Med. 313, 401-404. Gottsch, J.D., Pou, S., Bynoe, L.A., and Rosen, G.M. (1990) Invest. Ophthalmol. Vis. Sci. 31 :1674-l 682. Kaplan, B.H. (1983) Synthesis of Heme. In Hematology, 3rd edition, Chapter 31 (eds. Williams, Beutler, Erslev, Lichtman.) pp. 287-294, McGraw-Hill, New York. Buettner, G.R. and Oberly, L.W. (1979) FEBS Lett. 98, 18-20. Finkelstein, E., Rosen, G.M., and Rauckman, E.J. (1980) Arch. Biochem. Biophys. 200, l16. Janzen, E.G. (1980) A critical review of spin trapping in biological systems. In Free Radicals in Biology, vol. 4 (ed. W.A. Pryor) pp. 115-154, Academic Press, New York. Pou, S., Hassett, D.J., Britigan, B.E., Cohen, M.S., and Rosen, G.M. (1989) Anal. Biochem. 177, 1-6. Bennett, R., Brown, R.F.C., Clark, V.M., Sutherland, LO., and Todd, A. (1959) J. Cheryl. t&c. 2094-2102.
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