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.

10. 11.

12.

Poyer, J.L. and McCay, P.B. (1971) J. Biol. Chem. 246, 264-269. Chisolm Jr., J.J. and Brown, D.H. (1975) Clin. Chem. 21, 1669-1689. Buettner G.R., Oberley, L.W., and Leuthauser, S.W.H.C. (1978) Photochem.

Photobiol.

28,

693-695. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22.

Michiels, C. and Remacle, J. (1988) Eur. J. Biochem. 177, 435-441. Lynch, R.E. and Fridovich, I. (1978) J. Biol. Chem. 253, 4697-4699, Misra, H.P. and Fridovich, I. (1972) J. Biol. Chem. 247, 6960-6962. Hebbel, R.P., Eaton, J.W., Balasingam, M., and Steinberg, M.H. (1982) J. Clin. Invest. 70, 1253-l 259. Finkelstein, E., Rosen, G.M., and Rauckman, E.J. (1980) J. Amer. Chem. Sot. 102, 49944999. McCord, J.M. and Fridovich, I. (1969) J. Biol. Chem. 244, 6049-6055. Fee, J.A. and Valentine J.S. (1977) Chemical and physical properties of superoxide. fn Superoxide and Superoxide Dismutases feds. A.M. Michelson, J.M. McCord, and I. Fridovich) pp. 19-60, Academic Press, New York. Oehler, U.M. and Janzen. E.G. (1982) Can. J. Chem. 60, 1542-1548. Suttorp, N., Toepfer, W., and Roka, L. (1986) Am. J. Physiol. 251 (Cell. Physiol. ZO), C671 -C680. Freeman, B.A., and Crapo, J.D. (1982) Lab. Invest. 47. 412-426.

1310