[67]
PRODUCTION
OF OXYGEN
RADICALS
BY PHOTOSENSITIZATION
635
Role of GSH in Quinone-Induced Depletion of Total Cellular and Cytoskeletal Protein Thiols Intracellular GSH plays an important role in the defense against the toxicity of many xenobiotics including both alkylating and redox-active quinones. 1,3 In hepatocytes there is a clear relationship between quinoneinduced depletion of protein thiols and cytotoxicity. To investigate the effect of the intracellular glutathione level on the depletion of protein thiols triggered by quinone metabolism, GSH-depleted hepatocytes were incubated with menadione, p-benzoquinone, or 2,3-dimethoxy-l,4naphthoquinone, and both total and cytoskeletal protein thiols were assayed. As illustrated in Table I, GSH-depleted cells showed an enhanced susceptibility to quinone-induced depletion of both total and cytoskeletal protein thiols, in agreement with the enhanced toxicity and accelerated appearance of surface abnormalities observed under the same conditions. Conclusions The metabolism of cytotoxic levels of quinones in isolated hepatocytes is associated with the depletion of intracellular glutathione. This results from either alkylation or oxidation, depending on the quinone employed. During the metabolism of redox-active quinones, GSSG is formed and glutathione-protein mixed disulfides are detected in several subceUular fractions, including the cytoskeleton. Moreover, when glutathione reductase is inhibited, the accumulation of GSSG and mixed disulfides caused by low concentrations of the redox-active quinones is markedly potentiated. Thus, intracellular glutathione plays a critical role in the defense against quinone-associated cytotoxicity, particularly by preventing the quinone-induced modification of protein thiols, an effect that appears to be closely linked to the occurrence of irreversible cell damage.
[67] P r o d u c t i o n o f O x y g e n R a d i c a l s b y P h o t o s e n s i t i z a t i o n
By JOSEPH P. MARTIN, JR. and PAULA BURCH Introduction The cytotoxicity of illuminated photosensitizing agents in the presence of oxygen has long been recognized. It is commonly referred to as the METHODS IN ENZYMOLOGY, VOL. 186
Copyright © 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.
636
ORGAN, TISSUE, AND CELL DAMAGE
[67]
photodynamic e f f e c t . 1-4 A variety of compounds (e.g., synthetic dyes, antipsychotic drugs, antibiotics, and plant secondary compounds) exhibit this effect, and all groups of organisms are susceptible to photodamage. The types of cellular damage that occur include lipid peroxidation and membrane lysis, DNA base modification and DNA strand breakage, mutagenesis, and protein inactivation. L3-8 Dye-mediated photooxidations proceed by two different pathways. L2 In the first pathway (type I), excited triplet state dyes react directly with an oxidizable substrate. Electron or hydrogen atom transfer generates a semioxidized substrate radical and a semireduced dye radical. Subsequent reactions of both species with oxygen yield oxidized and modified substrates, regenerated ground state dye, oxygen radicals, and hydrogen peroxide (H202). When photooxidations occur within cells, damage may be caused during the primary oxidation events 3,4 or by further reactions of oxygen radicals and H202 with cell components.8-1° In the second pathway (type II), excited state dye triplets react directly with molecular oxygen, yielding ground state dye and singlet oxygen (~O2). Photodamage is caused by reactions of singlet oxygen with amino acids, nucleotides, and lipids? -6 Under conditions of high oxygen concentration, characteristic of organic solvents, and in the absence of strong reducing agents, the second pathway is favored by most photosensitizing compounds. Lz However, in aqueous buffer solutions, where the concentration of oxygen is low (0.20.25 mM), and in the presence of high reductant concentrations, the type I pathway will be favored. Suitable reductants in vitro include allylthiourea, ascorbate, aromatic amines, reduced glutathione, tetramethylethylenediamine and reduced pyridine nucleotides.9-~6 Within aerobic cells t C. S. Foote, in " F r e e Radicals in Biology" (W. Pryor, ed.) Vol. 2, p. 86. Academic Press, New York. 2 C. S. Foote, F. C. Shook, and R. B. Abakerli, this series Vol. 105, p. 36. J. D. Spikes and R. Livingston, Adv, Radiat. Biol. 3, 29 (1969). 4 j. D. Spikes and R. Straight, Annu. Rev. Phys. Chem. 18, 409 (1967). 5 N. Houba-Herin, C. M. Calberg-Bacq, J. Piette, and A. Van de Vorst, Photochem. Photobiol. 36, 297 (1982). 6 j. Piette, M. Lopez, C. M. Calberg-Bacq, and A. Van de Vorst, Int. J. Radiat. Biol. Relat. Stud. Phys. Chem. Med. 40, 427 (1981). 7 C. Wallis and H. L. Melnick, Photochem. Photobiol. 4, 159 (1965). 8 j. p. Martin, K. Colina, and N. Logsdon, J. Bacteriol. 169, 2516 (1987). 9 j. p. Martin and N. Logsdon, Arch. Biochem. Biophys. 2S6, 39 (1987). 10j. p. Martin and N. Logsdon, J. Biol. Chem./.62, 7213 (1987). u G. R. Beuttner, T. P. Doherty, and T. D. Bannister, Radiat. Environ. Biophys. 23, 235 (1984). 12 C. Beauchamp and I. Fridovich, Anal. Biochem. 44, 276 (1972).
[67]
PRODUCTIONOF OXYGENRADICALSBY PHOTOSENSITIZATION
637
the type I pathway may be favored because o f the low concentration o f free o x y g e n and the high intracellular levels of reductants such as reduced glutathione ( I - 6 mM) 17.1a and NAD(P)H (1 mM). 19 In this chapter we present methods o f generating superoxide, (O2-), hydrogen peroxide, and the hydroxyl radical (OH .) through photooxidations mediated by synthetic dyes both in vitro and in vivo, within the cytoplasm of E s c h e r i c h i a coll. I n Vitro Photooxidation Procedures D y e - M e d i a t e d G e n e r a t i o n o f 0 2 - . All reactions are carried out in 3 ml o f 50 m M potassium phosphate, 0.1 m M E D T A (pH 7.4-7.8). In the assay designed for 0 2 - detection the solution also contains 10 /zM oxidized c y t o c h r o m e c (Sigma type III). The reductant N A D H (nicotinamide adenine dinucleotide, reduced form) is added as a 20 m M stock solution made in the phosphate buffer at p H 7.0 and is diluted into the reaction mixture to a final concentration between 0.2 and 2.0 mM. Dye stock solutions o f 1 m M are also prepared in the potassium phosphate buffer using published extinction coefficients. 5 Reactions are carried out in fused silica cuvettes. N A D H depletion is monitored spectrophotometrically at 340 nm, and c y t o c h r o m e c reduction is followed at 550 nm. Superoxide generation is estimated by the inhibition of c y t o c h r o m e c reduction when Cu,Zn-SOD (copper, zinc-superoxide dismutase) is added to the reaction mixture. Cu,Zn-SOD is prepared as a 1 mg/ml stock solution in the potassium phosphate buffer. This corresponds to 3600 McCord and Fridovich units per ml. 2° SOD is added to the reaction to a final concentration of 20/zg/ml. Light incubation is done by setting cuvettes midway between two 20-W fluorescent lamps (GE F20.Tl2-pl) positioned 5 cm apart in a foillined box. Visible light intensity is measured with a Licor LI-185B radio m e t e r / p h o t o m e t e r using an LI-190SB cosine corrected quantum sensor.
13H. M. Hassan and I. Fridovich, Arch. Biochem. Biophys. 196, 385 (1979). ,4 I. Kraljic and L. Lindqvist, Photochem. Photobiol. 20, 351 (1974). 15G. Oster, J. S. Bellin, R. W. Kimball, and M. S. Schrader, J. Am. Chem. Soc. 81, 5095 (1959L 16A. H. Adelman and G. Oster, J. Am. Chem. Soc. 78, 3977 (1956). 17R° C. Fahey, W. C. Brown, W. B. Adams, and M. B. Worsham, J. Bacteriol. 133, 1126 (1978). 18j. T. Greenberg and B. Demple, J. Bacteriol. 168, 1026 (1986). 19K. B. Anderson and K. Von Meyenberg, J. Biol. Chem. 252, 4151 (1977). 2oj. M. McCord and I. Fridovich, J. Biol. Chem. 244, 6049 (1969).
638
ORGAN, TISSUE, AND CELL DAMAGE
[671
Reactions are started by addition of dye to the desired concentration followed by illumination. Cuvettes are removed from the light at 1-min intervals and examined spectrophotometrically at either 340 nm or 550 nm in a UV-visible spectrophotometer. Hydroxyl Radical Generation. The hydroxyl radical is generated in the system described above when the reaction mixture is supplemented with 50/xM ferric chloride, added as a 5 mM acidic stock solution. Also, the buffer concentration is increased to 0.15 M potassium phosphate (pH 7.6) and is supplemented with 4 mM sodium salicylate. Reactions are started as described above and are incubated for 1 hr, after which the degree of salicylate hydroxylation is determined by the method of Halliwell. 2~ The hydroxylated diphenolic products are extracted from the reaction mixture with chilled diethyl ether, and the concentration of diphenol is estimated colorimetrically at 510 nm. 2,3-Dihydroxybenzoate is used as a calibration standard and is similarly extracted from mock reaction mixtures that do not contain N A D H or salicylate.
Results Dye-Mediated Cytochrome c Reduction. Figure 1 shows the photosensitized reduction of cytochrome c by the naphthalimide dye lucifer yellow CH. This dye is commonly used to stain and ablate subcellular structures in laser microsurgery. 22,z3 Cytochrome c reduction rates are proportional to the dye concentration and are negligible in the absence of dye, reductant, or light. Figure 1B illustrates the inhibitory effect of Cu,Zn-SOD. Cytochrome c reduction rates are diminished as the SOD concentration in the reaction mixture is increased, but equivalent concentrations of bovine serum albumin (BSA) are without effect on the reaction rate. N A D H oxidation parallels cytochrome c reduction (Fig. 2A). When various dye classes are compared it is clear that thiazine dyes (i.e., azure C, methylene blue, toluidine blue O) are the most reactive sensitizers under the specified illumination conditions. Salicylate Hydroxylation by Dye-Mediated Hydroxyl Radical Generation. Figure 2B illustrates that dye-sensitized photooxidation of N A D H yields the hydroxyl radical. The relative abilities of the dyes to sensitize the hydroxylation of salicylate correspond to their relative abilities to oxidize N A D H under the illumination conditions employed. Hydroxylation rates also depend on the intensity of illumination and on the dye concentration. Although many acridines (e.g., proflavin, acridine orange) are potent photosensitizing agents, the substituted acridine dye quina21 B. Halliwell, FEBS Lett. 92, 321 (1978). 22 W. W. Stewart, Cell 14, 741 (1978). 23 j. p. Miller and A. I. Selverston, Science 206, 702 (1979).
0.25
A
0.20
Ec: 0.15 o
2
3
J/
0.10
(
0.05
0.00
5
0
0.25
10
15
2O
25
B 1
0.20
2
E 0.15 o=
3
0.10
0.05
5
0.00 0
5
10 15 Minutes
20
25
FIG. 1. Lucifer yellow CH-sensitized cytochrome c reduction by NADH and its inhibition by superoxide dismutase (SOD). (A) Cytochrome c reduction was monitored as a function of lucifer yellow CH concentration. Assays contained 3 ml of 50 mM potassium phosphate, 0.1 mM EDTA (pH 7.8 at 25°). Line 1, 47 p.M; line 2, 27/aA/; line 3, 13/~M; line 4, 10/LM; line 5, 6.6/LM lucifer yellow CH; line 6, NADH, no lucifer yellow CH; line 7, 47 /LM lucifer yellow CH, no NADH. (B) Lucifer yellow CH, 27 p.M, was incubated with BSA or increasing concentrations of SOD. Line 1, 27/.¢M lucifer yellow CH, light; line 2, plus 30 /~g BSA; line 3, plus 2 units SOD; line 4, plus 4 units SOD; line 5, plus 24 units SOD; line 6, plus 24 units SOD, dark incubation. [From J. P. Martin and N. Logsdon, Photochem. Photobiol. 46, 45 (1987).]
640
[67]
ORGAN, TISSUE, AND CELL DAMAGE
crine does not sensitize either NADH oxidation or salicylate hydroxylation. Hydroxyl radical production by all dyes is completely inhibited by the addition of catalase, by the addition of the hydroxyl radical scavenger thiourea, or by the substitution of desferrioxamine for EDTA in the reaction mixture. 2.0'
1.5
o
1.0
,~4
0.5
0.0 ~
0
2
4
6
8
10
12
Minutes FIG. 2. (A) NADI-I oxidation mediated by phototoxic dyes. Oxidation of NADH was observed as a decrease in absorbance at 340 nm. Solutions contained 0.32 mM NADH in 3 ml of 50 mM potassium phosphate, 0.1 mM EDTA (pH 7.8 at 25°), and dyes at the indicated concentrations. These were illuminated with visible light, 4.6 mW/cm 2. Line 1, 7/~M azure C; line 2, 7/~M toluidine blue O; line 3, 5 / , M methylene blue; line 4, 5 I~M rose bengal; line 5, 5/xM acriflavin; line 6, 10/zM neutral red; line 7, 5/zM proflavin; line 8, 10/~M erythrosine; line 9, 10 wM pyronin; line 10, no dye addition. (B) Hydroxyl radical generation by dyemediated NADH photooxidation. Solutions contained 0.15 M potassium phosphate (pH 7.6), 1.0 mM NADH, 4.0 mM sodium salicylate, 50 p~M Fe-EDTA, and dyes (azure C, [:]; proflavin, I1; neutral red, x ; and quinacrine, O) at the indicated concentrations. Hydroxyl radical production was estimated as the hydroxylation of salicylate. [From J. P. Martin and P. Burch, in "Oxy-Radicals in Molecular Biology and Pathology" (N. Cerutti, J. McCord, and I. Fridovich, eds.), p. 394. Alan R. Liss, New York, 1988.]
[67]
PRODUCTION OF OXYGEN RADICALS BY PHOTOSENSITIZATION
641
150 D
125
/.
tlJ .d
~oI00 z ..I
o a 75
1
n..i x
o " 50
25
2
4
6
50
DYE CONCENTRATION (l.tM)
FIc. 2. (continued)
In Vivo Photooxidation Procedures lntracellular Generation o f Oxygen Radicals and SOD Induction Mediated by Synthetic Dyes in Escherichia coli. Escherichia coli B (ATCC 23226) may be obtained from the American Type Culture Collection. Cultures are grown in M9 salts plus 0.4% glucose (Na2HPO4, 3 g; NaCI, 0.5 g; NH4CI, 1 g; MgSO4, 495 mg; CaCI2, I0.1 mg; glucose, 4.0 g per liter) or in nutrient broth (NB). Media are titrated to pH 7.2. Bacteria are grown in 50-ml cultures in 250-ml Nephelo flasks at 37° with vigorous shaking (200 rpm) in New Brunswick G-76 shakers. Growth is followed using a Klett-Summerson colorimeter.
642
ORGAN, TISSUE, AND CELL DAMAGE
[67l
Preinduction of E. coli manganese superoxide dismutase (Mn-SOD) and catalase to levels 10- to 20-fold greater than the basal levels is achieved by the addition of 100/.~M manganese and 10/zM paraquat to cultures 1 hr after inoculation into M9 salts plus glucose. 24 The inoculum is 2%, and the cells are from a stationary phase overnight culture grown in M9. Growth in the presence of paraquat and manganese is allowed to continue for 3-5 hr prior to harvest. SOD induction experiments are carried out by inoculating 50 ml of NB containing dye at the desired concentration to 2% with an overnight culture of E. coli B grown in NB. Following inoculation, growth is continued for 6 hr at 37°, 200 rpm in a gyrorotary shaker. Ceils are grown either in complete darkness or under a 150-W GE reflector flood lamp enclosed within an aluminum foil tent. The light intensity at the surface of the broth should be 1.9-2.2 mW/cm 2. Bacterial cells are prepared for superoxide dismutase and catalase assays by centrifugation at 7000 g at 4° for 20 min followed by washing 2 times with M9 salts (pH 7.4). The cells are resuspended in 2 ml of 50 mM potassium phosphate, 0.1 mM EDTA (pH 7.4) and are lysed by sonication in a Heat Systems-Ultrasonics Model 370 sonicator. Sonication is done in a cup horn at 100 W at 4° in six 1-min bursts. Lysates are centrifuged at 15,000 g for 20 min at 4°, and the supernatants are dialyzed for 48 hr against three 400-volume changes of 10 mM potassium phosphate, 0.1 mM EDTA (pH 7.4). Superoxide dismutase is assayed by the xanthine oxidase-cytochrome c method. 2° Xanthine oxidase is purified from unpasteurized cream. 25 Catalase is estimated according to Beers and Sizer. 26 Protein is determined by the Bradford assay 27 using bovine ~/-globulin as a standard. Estimation of Dye-Mediated Lethality in Escherichia coli B. In order to determine lethality, fresh cultures are started with a 2% inoculum of cells grown overnight in M9 salts plus glucose. Cultures are harvested from the mid-log phase of growth by centrifugation at 7000 g for 20 min at 4°. After 2 washes with M9 salts (pH 7.4), cells are resuspended in 3 ml of M9 plus glucose, 80/xg/ml chloramphenicol (pH 7.4) in presterilized cuvettes. Final cell density is 0.5-1 × 108 cells/ml. Hydroxyl radical scavengers are added to the incubation medium prior to cell addition. Filtersterilized superoxide dismutase, catalase, bovine serum albumin, and 24 S. Y. R. Pugh and I. Fridovich, J. Bacteriol. 162, 196 (1985). z5 W. O. Waud, F. O. Brady, R. D. Wiley, and K. V. Rajagopalan, Arch. Biochem. Biophys. 169, 695 (1975). z6 R. F. Beers and I. W. Sizer, J. Biol. Chem. 195, 133 (1952). 27 M. Bradford, Anal. Biochem. 72, 248 (1976).
[67]
PRODUCTION OF OXYGEN RADICALS BY PHOTOSENSITIZATION
643
100~
80
6o
40
20
i
0
|
I
I
I
l
2
4
6
8
10
12
Toluidine Blue (uM) F[o. 3. Induction of SOD in E. coil B mediated by toluidine blue O plus light. Cells were grown in NB with toluidine blue O at the indicated concentrations. After 6 hr of growth, cells were harvested and lysed, and the extracts were assayed for SOD as described in the text. I7, Cells grown under illumination; I1, cells grown in darkness. [From J. P. Martin and N. Logsdon, Arch. Biochem. Biophys. 256, 39 (1978).]
dyes are added after the addition of cells. Anaerobic incubations are carried out in anaerobic quartz cuvettes. 28 The incubation solutions are scrubbed for 25 min with ultrapure nitrogen prior to sealing the cuvettes and tipping in the photosensitizer from a side arm. All treatment cuvettes are periodically inverted during incubation to assure adequate oxygenation and uniform cell suspensions. Treated cells are diluted into sterile M9 salts (pH 7.4) and incubated for 20 min at 23 ° to allow diffusion of the dyes from cells prior to plating. Surviving cells are plated on Luria broth (LB) medium solidified with 1.8% Bacto-agar at three dilutions and in triplicate. Plates are incubated at 37° in the dark for 16-24 hr and then counted. E. K. Hodgson, J. M. McCord, and I. Fridovich, Anal. Biochem. 5, 470 (1973).
644
[67]
ORGAN, TISSUE, AND CELL DAMAGE
Results D y e - M e d i a t e d S O D Induction in Escherichia coli B. D y e - m e d i a t e d phototoxicity by oxygen radicals involves reduced dye intermediates. D y e r e d u c t i o n b y i n t r a c e l l u l a r s u b s t r a t e s will b e o b s e r v e d in vivo o n l y if t h e d y e s p e n e t r a t e t h e b a c t e r i a l cell m e m b r a n e s . L o w m o l e c u l a r w e i g h t c a t i o n i c d y e s s u c h as t h e t h i a z i n e s a n d a c r i d i n e s s h o u l d e a s i l y p a s s
10 =
101
100 a ==
.> =1
O3
10 "1
O.
10 "2
10 .3
1 0 .4
,
0
I
20
~
I
40
I
I
60
80
Minutes
FIG. 4. Protection against azure C phototoxicity conferred by induction of Mn-SOD and catalase, and by addition of oxygen radical scavengers. Incubation conditions were as described in the text. Mn-SOD and catalase levels were elevated in E. coli B by addition of 10/.~M paraquat and 100 p,M Mn to the growth medium. Cell homogenates were assayed for SOD and catalase activities. Line 1, 2/zM azure C, 10.5 U/mg SOD, 0.5 U/mg catalase; line 2, 2 p.M azure C, 161 U/rag SOD, 2.2 U/rag catalase; line 3, 2/~M azure C, 161 U/rag SOD, 2.2 U/rag catalase, 300 units exogenous SOD, 3000 units exogenous catalase; line 4, 2 #.M azure C, 10.5 U/mg SOD, 0.5 U/rag catalase, 500 mM dimethyl sulfoxide, 300 units exogenous SOD, 3000 units exogenous catalase; line 5, 2/xM azure C, 161 U/mg SOD, 2.2 U/mg catalase, 500 ram dimethyl sulfoxide, 300 units exogenous SOD, 3000 units exogenous catalase. [From J. P. Martin and N. Logsdon, J. Biol. Chem. 262, 7213 (1987).]
[67]
PRODUCTION OF OXYGEN RADICALS BY PHOTOSENSITIZATION
645
through the porin channels of the E. coli outer membrane 29 and are readily taken up by the cells at neutral pH. 3° On the other hand, the passage of anionic compounds through porin channels is retarded, 3~ and significant accumulation of anionic xanthene dyes by E. coli occurs only under acidic conditions) ° Escherichia coli is able to induce SOD and overcome the oxidative stress presented by redox-active compounds. 32,33Analysis of SOD levels in the cultures grown at increasing concentrations of the photosensitizing dye toluidine blue O (Fig. 3) reveals that there is a greater than 4-fold induction of superoxide dismutase at the highest concentration of this thiazine dye. Growth in the dark at high dye concentrations produces a more modest induction. Dye-Mediated Cell Lethality. Treatment of E. coli B with the thiazine dye azure C is lethal. Lethality is dependent on light and oxygen. Bacteria in dark incubations are unaffected by azure C, and anaerobic incubations are only mildly toxic (data not shown). The lethality observed in glucose minimal medium containing chloramphenicol is probably enhanced by the inability of E. coli B to induce superoxide dismutase under these conditions. The toxicity of azure C is relieved by the hydroxyl radical scavenger dimethylsulfoxide and the H202 scavenger catalase (Fig. 4). Furthermore, high intracellular levels of superoxide dismutase and catalase also relieve azure C toxicity. Almost complete protection is obtained by addition of dimethyl sulfoxide and exogenous Cu,Zn-SOD and catalase to E. coli B containing elevated intraceUular levels of superoxide dismutase and catalase (Fig. 4). Acknowledgments This work was supported by Public Health Service Research Grants AI-19695 and GM-07833, Grant C-900from the Robert A. Welch Foundation, and a grant from the American Heart Association Texas Affiliate.
29 H. Nikaido and E. Y. Rosenberg, J. Bacteriol. 153, 241 (1983). 3o j. S. Bellin, L. Lutwick, and B. Jonas, Arch. Biochem. Biophys. 132, 157 0969). 31 H. Nikaido, E. Y. Rosenberg, and J. Foulds, J. Bacteriol. 153, 232 (1983). 32 H. M. Hassan and I. Fridovich, J. Bacteriol. 141, 156 0980). 33 H. M. Hassan and I. Fridovich, J. Biol. Chem. 2,52, 7667 (1977).
PRODUCTION
OF OXYGEN
RADICALS
BY PHOTOSENSITIZATION
635
Role of GSH in Quinone-Induced Depletion of Total Cellular and Cytoskeletal Protein Thiols Intracellular GSH plays an important role in the defense against the toxicity of many xenobiotics including both alkylating and redox-active quinones. 1,3 In hepatocytes there is a clear relationship between quinoneinduced depletion of protein thiols and cytotoxicity. To investigate the effect of the intracellular glutathione level on the depletion of protein thiols triggered by quinone metabolism, GSH-depleted hepatocytes were incubated with menadione, p-benzoquinone, or 2,3-dimethoxy-l,4naphthoquinone, and both total and cytoskeletal protein thiols were assayed. As illustrated in Table I, GSH-depleted cells showed an enhanced susceptibility to quinone-induced depletion of both total and cytoskeletal protein thiols, in agreement with the enhanced toxicity and accelerated appearance of surface abnormalities observed under the same conditions. Conclusions The metabolism of cytotoxic levels of quinones in isolated hepatocytes is associated with the depletion of intracellular glutathione. This results from either alkylation or oxidation, depending on the quinone employed. During the metabolism of redox-active quinones, GSSG is formed and glutathione-protein mixed disulfides are detected in several subceUular fractions, including the cytoskeleton. Moreover, when glutathione reductase is inhibited, the accumulation of GSSG and mixed disulfides caused by low concentrations of the redox-active quinones is markedly potentiated. Thus, intracellular glutathione plays a critical role in the defense against quinone-associated cytotoxicity, particularly by preventing the quinone-induced modification of protein thiols, an effect that appears to be closely linked to the occurrence of irreversible cell damage.
[67] P r o d u c t i o n o f O x y g e n R a d i c a l s b y P h o t o s e n s i t i z a t i o n
By JOSEPH P. MARTIN, JR. and PAULA BURCH Introduction The cytotoxicity of illuminated photosensitizing agents in the presence of oxygen has long been recognized. It is commonly referred to as the METHODS IN ENZYMOLOGY, VOL. 186
Copyright © 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.
636
ORGAN, TISSUE, AND CELL DAMAGE
[67]
photodynamic e f f e c t . 1-4 A variety of compounds (e.g., synthetic dyes, antipsychotic drugs, antibiotics, and plant secondary compounds) exhibit this effect, and all groups of organisms are susceptible to photodamage. The types of cellular damage that occur include lipid peroxidation and membrane lysis, DNA base modification and DNA strand breakage, mutagenesis, and protein inactivation. L3-8 Dye-mediated photooxidations proceed by two different pathways. L2 In the first pathway (type I), excited triplet state dyes react directly with an oxidizable substrate. Electron or hydrogen atom transfer generates a semioxidized substrate radical and a semireduced dye radical. Subsequent reactions of both species with oxygen yield oxidized and modified substrates, regenerated ground state dye, oxygen radicals, and hydrogen peroxide (H202). When photooxidations occur within cells, damage may be caused during the primary oxidation events 3,4 or by further reactions of oxygen radicals and H202 with cell components.8-1° In the second pathway (type II), excited state dye triplets react directly with molecular oxygen, yielding ground state dye and singlet oxygen (~O2). Photodamage is caused by reactions of singlet oxygen with amino acids, nucleotides, and lipids? -6 Under conditions of high oxygen concentration, characteristic of organic solvents, and in the absence of strong reducing agents, the second pathway is favored by most photosensitizing compounds. Lz However, in aqueous buffer solutions, where the concentration of oxygen is low (0.20.25 mM), and in the presence of high reductant concentrations, the type I pathway will be favored. Suitable reductants in vitro include allylthiourea, ascorbate, aromatic amines, reduced glutathione, tetramethylethylenediamine and reduced pyridine nucleotides.9-~6 Within aerobic cells t C. S. Foote, in " F r e e Radicals in Biology" (W. Pryor, ed.) Vol. 2, p. 86. Academic Press, New York. 2 C. S. Foote, F. C. Shook, and R. B. Abakerli, this series Vol. 105, p. 36. J. D. Spikes and R. Livingston, Adv, Radiat. Biol. 3, 29 (1969). 4 j. D. Spikes and R. Straight, Annu. Rev. Phys. Chem. 18, 409 (1967). 5 N. Houba-Herin, C. M. Calberg-Bacq, J. Piette, and A. Van de Vorst, Photochem. Photobiol. 36, 297 (1982). 6 j. Piette, M. Lopez, C. M. Calberg-Bacq, and A. Van de Vorst, Int. J. Radiat. Biol. Relat. Stud. Phys. Chem. Med. 40, 427 (1981). 7 C. Wallis and H. L. Melnick, Photochem. Photobiol. 4, 159 (1965). 8 j. p. Martin, K. Colina, and N. Logsdon, J. Bacteriol. 169, 2516 (1987). 9 j. p. Martin and N. Logsdon, Arch. Biochem. Biophys. 2S6, 39 (1987). 10j. p. Martin and N. Logsdon, J. Biol. Chem./.62, 7213 (1987). u G. R. Beuttner, T. P. Doherty, and T. D. Bannister, Radiat. Environ. Biophys. 23, 235 (1984). 12 C. Beauchamp and I. Fridovich, Anal. Biochem. 44, 276 (1972).
[67]
PRODUCTIONOF OXYGENRADICALSBY PHOTOSENSITIZATION
637
the type I pathway may be favored because o f the low concentration o f free o x y g e n and the high intracellular levels of reductants such as reduced glutathione ( I - 6 mM) 17.1a and NAD(P)H (1 mM). 19 In this chapter we present methods o f generating superoxide, (O2-), hydrogen peroxide, and the hydroxyl radical (OH .) through photooxidations mediated by synthetic dyes both in vitro and in vivo, within the cytoplasm of E s c h e r i c h i a coll. I n Vitro Photooxidation Procedures D y e - M e d i a t e d G e n e r a t i o n o f 0 2 - . All reactions are carried out in 3 ml o f 50 m M potassium phosphate, 0.1 m M E D T A (pH 7.4-7.8). In the assay designed for 0 2 - detection the solution also contains 10 /zM oxidized c y t o c h r o m e c (Sigma type III). The reductant N A D H (nicotinamide adenine dinucleotide, reduced form) is added as a 20 m M stock solution made in the phosphate buffer at p H 7.0 and is diluted into the reaction mixture to a final concentration between 0.2 and 2.0 mM. Dye stock solutions o f 1 m M are also prepared in the potassium phosphate buffer using published extinction coefficients. 5 Reactions are carried out in fused silica cuvettes. N A D H depletion is monitored spectrophotometrically at 340 nm, and c y t o c h r o m e c reduction is followed at 550 nm. Superoxide generation is estimated by the inhibition of c y t o c h r o m e c reduction when Cu,Zn-SOD (copper, zinc-superoxide dismutase) is added to the reaction mixture. Cu,Zn-SOD is prepared as a 1 mg/ml stock solution in the potassium phosphate buffer. This corresponds to 3600 McCord and Fridovich units per ml. 2° SOD is added to the reaction to a final concentration of 20/zg/ml. Light incubation is done by setting cuvettes midway between two 20-W fluorescent lamps (GE F20.Tl2-pl) positioned 5 cm apart in a foillined box. Visible light intensity is measured with a Licor LI-185B radio m e t e r / p h o t o m e t e r using an LI-190SB cosine corrected quantum sensor.
13H. M. Hassan and I. Fridovich, Arch. Biochem. Biophys. 196, 385 (1979). ,4 I. Kraljic and L. Lindqvist, Photochem. Photobiol. 20, 351 (1974). 15G. Oster, J. S. Bellin, R. W. Kimball, and M. S. Schrader, J. Am. Chem. Soc. 81, 5095 (1959L 16A. H. Adelman and G. Oster, J. Am. Chem. Soc. 78, 3977 (1956). 17R° C. Fahey, W. C. Brown, W. B. Adams, and M. B. Worsham, J. Bacteriol. 133, 1126 (1978). 18j. T. Greenberg and B. Demple, J. Bacteriol. 168, 1026 (1986). 19K. B. Anderson and K. Von Meyenberg, J. Biol. Chem. 252, 4151 (1977). 2oj. M. McCord and I. Fridovich, J. Biol. Chem. 244, 6049 (1969).
638
ORGAN, TISSUE, AND CELL DAMAGE
[671
Reactions are started by addition of dye to the desired concentration followed by illumination. Cuvettes are removed from the light at 1-min intervals and examined spectrophotometrically at either 340 nm or 550 nm in a UV-visible spectrophotometer. Hydroxyl Radical Generation. The hydroxyl radical is generated in the system described above when the reaction mixture is supplemented with 50/xM ferric chloride, added as a 5 mM acidic stock solution. Also, the buffer concentration is increased to 0.15 M potassium phosphate (pH 7.6) and is supplemented with 4 mM sodium salicylate. Reactions are started as described above and are incubated for 1 hr, after which the degree of salicylate hydroxylation is determined by the method of Halliwell. 2~ The hydroxylated diphenolic products are extracted from the reaction mixture with chilled diethyl ether, and the concentration of diphenol is estimated colorimetrically at 510 nm. 2,3-Dihydroxybenzoate is used as a calibration standard and is similarly extracted from mock reaction mixtures that do not contain N A D H or salicylate.
Results Dye-Mediated Cytochrome c Reduction. Figure 1 shows the photosensitized reduction of cytochrome c by the naphthalimide dye lucifer yellow CH. This dye is commonly used to stain and ablate subcellular structures in laser microsurgery. 22,z3 Cytochrome c reduction rates are proportional to the dye concentration and are negligible in the absence of dye, reductant, or light. Figure 1B illustrates the inhibitory effect of Cu,Zn-SOD. Cytochrome c reduction rates are diminished as the SOD concentration in the reaction mixture is increased, but equivalent concentrations of bovine serum albumin (BSA) are without effect on the reaction rate. N A D H oxidation parallels cytochrome c reduction (Fig. 2A). When various dye classes are compared it is clear that thiazine dyes (i.e., azure C, methylene blue, toluidine blue O) are the most reactive sensitizers under the specified illumination conditions. Salicylate Hydroxylation by Dye-Mediated Hydroxyl Radical Generation. Figure 2B illustrates that dye-sensitized photooxidation of N A D H yields the hydroxyl radical. The relative abilities of the dyes to sensitize the hydroxylation of salicylate correspond to their relative abilities to oxidize N A D H under the illumination conditions employed. Hydroxylation rates also depend on the intensity of illumination and on the dye concentration. Although many acridines (e.g., proflavin, acridine orange) are potent photosensitizing agents, the substituted acridine dye quina21 B. Halliwell, FEBS Lett. 92, 321 (1978). 22 W. W. Stewart, Cell 14, 741 (1978). 23 j. p. Miller and A. I. Selverston, Science 206, 702 (1979).
0.25
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FIG. 1. Lucifer yellow CH-sensitized cytochrome c reduction by NADH and its inhibition by superoxide dismutase (SOD). (A) Cytochrome c reduction was monitored as a function of lucifer yellow CH concentration. Assays contained 3 ml of 50 mM potassium phosphate, 0.1 mM EDTA (pH 7.8 at 25°). Line 1, 47 p.M; line 2, 27/aA/; line 3, 13/~M; line 4, 10/LM; line 5, 6.6/LM lucifer yellow CH; line 6, NADH, no lucifer yellow CH; line 7, 47 /LM lucifer yellow CH, no NADH. (B) Lucifer yellow CH, 27 p.M, was incubated with BSA or increasing concentrations of SOD. Line 1, 27/.¢M lucifer yellow CH, light; line 2, plus 30 /~g BSA; line 3, plus 2 units SOD; line 4, plus 4 units SOD; line 5, plus 24 units SOD; line 6, plus 24 units SOD, dark incubation. [From J. P. Martin and N. Logsdon, Photochem. Photobiol. 46, 45 (1987).]
640
[67]
ORGAN, TISSUE, AND CELL DAMAGE
crine does not sensitize either NADH oxidation or salicylate hydroxylation. Hydroxyl radical production by all dyes is completely inhibited by the addition of catalase, by the addition of the hydroxyl radical scavenger thiourea, or by the substitution of desferrioxamine for EDTA in the reaction mixture. 2.0'
1.5
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Minutes FIG. 2. (A) NADI-I oxidation mediated by phototoxic dyes. Oxidation of NADH was observed as a decrease in absorbance at 340 nm. Solutions contained 0.32 mM NADH in 3 ml of 50 mM potassium phosphate, 0.1 mM EDTA (pH 7.8 at 25°), and dyes at the indicated concentrations. These were illuminated with visible light, 4.6 mW/cm 2. Line 1, 7/~M azure C; line 2, 7/~M toluidine blue O; line 3, 5 / , M methylene blue; line 4, 5 I~M rose bengal; line 5, 5/xM acriflavin; line 6, 10/zM neutral red; line 7, 5/zM proflavin; line 8, 10/~M erythrosine; line 9, 10 wM pyronin; line 10, no dye addition. (B) Hydroxyl radical generation by dyemediated NADH photooxidation. Solutions contained 0.15 M potassium phosphate (pH 7.6), 1.0 mM NADH, 4.0 mM sodium salicylate, 50 p~M Fe-EDTA, and dyes (azure C, [:]; proflavin, I1; neutral red, x ; and quinacrine, O) at the indicated concentrations. Hydroxyl radical production was estimated as the hydroxylation of salicylate. [From J. P. Martin and P. Burch, in "Oxy-Radicals in Molecular Biology and Pathology" (N. Cerutti, J. McCord, and I. Fridovich, eds.), p. 394. Alan R. Liss, New York, 1988.]
[67]
PRODUCTION OF OXYGEN RADICALS BY PHOTOSENSITIZATION
641
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FIc. 2. (continued)
In Vivo Photooxidation Procedures lntracellular Generation o f Oxygen Radicals and SOD Induction Mediated by Synthetic Dyes in Escherichia coli. Escherichia coli B (ATCC 23226) may be obtained from the American Type Culture Collection. Cultures are grown in M9 salts plus 0.4% glucose (Na2HPO4, 3 g; NaCI, 0.5 g; NH4CI, 1 g; MgSO4, 495 mg; CaCI2, I0.1 mg; glucose, 4.0 g per liter) or in nutrient broth (NB). Media are titrated to pH 7.2. Bacteria are grown in 50-ml cultures in 250-ml Nephelo flasks at 37° with vigorous shaking (200 rpm) in New Brunswick G-76 shakers. Growth is followed using a Klett-Summerson colorimeter.
642
ORGAN, TISSUE, AND CELL DAMAGE
[67l
Preinduction of E. coli manganese superoxide dismutase (Mn-SOD) and catalase to levels 10- to 20-fold greater than the basal levels is achieved by the addition of 100/.~M manganese and 10/zM paraquat to cultures 1 hr after inoculation into M9 salts plus glucose. 24 The inoculum is 2%, and the cells are from a stationary phase overnight culture grown in M9. Growth in the presence of paraquat and manganese is allowed to continue for 3-5 hr prior to harvest. SOD induction experiments are carried out by inoculating 50 ml of NB containing dye at the desired concentration to 2% with an overnight culture of E. coli B grown in NB. Following inoculation, growth is continued for 6 hr at 37°, 200 rpm in a gyrorotary shaker. Ceils are grown either in complete darkness or under a 150-W GE reflector flood lamp enclosed within an aluminum foil tent. The light intensity at the surface of the broth should be 1.9-2.2 mW/cm 2. Bacterial cells are prepared for superoxide dismutase and catalase assays by centrifugation at 7000 g at 4° for 20 min followed by washing 2 times with M9 salts (pH 7.4). The cells are resuspended in 2 ml of 50 mM potassium phosphate, 0.1 mM EDTA (pH 7.4) and are lysed by sonication in a Heat Systems-Ultrasonics Model 370 sonicator. Sonication is done in a cup horn at 100 W at 4° in six 1-min bursts. Lysates are centrifuged at 15,000 g for 20 min at 4°, and the supernatants are dialyzed for 48 hr against three 400-volume changes of 10 mM potassium phosphate, 0.1 mM EDTA (pH 7.4). Superoxide dismutase is assayed by the xanthine oxidase-cytochrome c method. 2° Xanthine oxidase is purified from unpasteurized cream. 25 Catalase is estimated according to Beers and Sizer. 26 Protein is determined by the Bradford assay 27 using bovine ~/-globulin as a standard. Estimation of Dye-Mediated Lethality in Escherichia coli B. In order to determine lethality, fresh cultures are started with a 2% inoculum of cells grown overnight in M9 salts plus glucose. Cultures are harvested from the mid-log phase of growth by centrifugation at 7000 g for 20 min at 4°. After 2 washes with M9 salts (pH 7.4), cells are resuspended in 3 ml of M9 plus glucose, 80/xg/ml chloramphenicol (pH 7.4) in presterilized cuvettes. Final cell density is 0.5-1 × 108 cells/ml. Hydroxyl radical scavengers are added to the incubation medium prior to cell addition. Filtersterilized superoxide dismutase, catalase, bovine serum albumin, and 24 S. Y. R. Pugh and I. Fridovich, J. Bacteriol. 162, 196 (1985). z5 W. O. Waud, F. O. Brady, R. D. Wiley, and K. V. Rajagopalan, Arch. Biochem. Biophys. 169, 695 (1975). z6 R. F. Beers and I. W. Sizer, J. Biol. Chem. 195, 133 (1952). 27 M. Bradford, Anal. Biochem. 72, 248 (1976).
[67]
PRODUCTION OF OXYGEN RADICALS BY PHOTOSENSITIZATION
643
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Toluidine Blue (uM) F[o. 3. Induction of SOD in E. coil B mediated by toluidine blue O plus light. Cells were grown in NB with toluidine blue O at the indicated concentrations. After 6 hr of growth, cells were harvested and lysed, and the extracts were assayed for SOD as described in the text. I7, Cells grown under illumination; I1, cells grown in darkness. [From J. P. Martin and N. Logsdon, Arch. Biochem. Biophys. 256, 39 (1978).]
dyes are added after the addition of cells. Anaerobic incubations are carried out in anaerobic quartz cuvettes. 28 The incubation solutions are scrubbed for 25 min with ultrapure nitrogen prior to sealing the cuvettes and tipping in the photosensitizer from a side arm. All treatment cuvettes are periodically inverted during incubation to assure adequate oxygenation and uniform cell suspensions. Treated cells are diluted into sterile M9 salts (pH 7.4) and incubated for 20 min at 23 ° to allow diffusion of the dyes from cells prior to plating. Surviving cells are plated on Luria broth (LB) medium solidified with 1.8% Bacto-agar at three dilutions and in triplicate. Plates are incubated at 37° in the dark for 16-24 hr and then counted. E. K. Hodgson, J. M. McCord, and I. Fridovich, Anal. Biochem. 5, 470 (1973).
644
[67]
ORGAN, TISSUE, AND CELL DAMAGE
Results D y e - M e d i a t e d S O D Induction in Escherichia coli B. D y e - m e d i a t e d phototoxicity by oxygen radicals involves reduced dye intermediates. D y e r e d u c t i o n b y i n t r a c e l l u l a r s u b s t r a t e s will b e o b s e r v e d in vivo o n l y if t h e d y e s p e n e t r a t e t h e b a c t e r i a l cell m e m b r a n e s . L o w m o l e c u l a r w e i g h t c a t i o n i c d y e s s u c h as t h e t h i a z i n e s a n d a c r i d i n e s s h o u l d e a s i l y p a s s
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FIG. 4. Protection against azure C phototoxicity conferred by induction of Mn-SOD and catalase, and by addition of oxygen radical scavengers. Incubation conditions were as described in the text. Mn-SOD and catalase levels were elevated in E. coli B by addition of 10/.~M paraquat and 100 p,M Mn to the growth medium. Cell homogenates were assayed for SOD and catalase activities. Line 1, 2/zM azure C, 10.5 U/mg SOD, 0.5 U/mg catalase; line 2, 2 p.M azure C, 161 U/rag SOD, 2.2 U/rag catalase; line 3, 2/~M azure C, 161 U/rag SOD, 2.2 U/rag catalase, 300 units exogenous SOD, 3000 units exogenous catalase; line 4, 2 #.M azure C, 10.5 U/mg SOD, 0.5 U/rag catalase, 500 mM dimethyl sulfoxide, 300 units exogenous SOD, 3000 units exogenous catalase; line 5, 2/xM azure C, 161 U/mg SOD, 2.2 U/mg catalase, 500 ram dimethyl sulfoxide, 300 units exogenous SOD, 3000 units exogenous catalase. [From J. P. Martin and N. Logsdon, J. Biol. Chem. 262, 7213 (1987).]
[67]
PRODUCTION OF OXYGEN RADICALS BY PHOTOSENSITIZATION
645
through the porin channels of the E. coli outer membrane 29 and are readily taken up by the cells at neutral pH. 3° On the other hand, the passage of anionic compounds through porin channels is retarded, 3~ and significant accumulation of anionic xanthene dyes by E. coli occurs only under acidic conditions) ° Escherichia coli is able to induce SOD and overcome the oxidative stress presented by redox-active compounds. 32,33Analysis of SOD levels in the cultures grown at increasing concentrations of the photosensitizing dye toluidine blue O (Fig. 3) reveals that there is a greater than 4-fold induction of superoxide dismutase at the highest concentration of this thiazine dye. Growth in the dark at high dye concentrations produces a more modest induction. Dye-Mediated Cell Lethality. Treatment of E. coli B with the thiazine dye azure C is lethal. Lethality is dependent on light and oxygen. Bacteria in dark incubations are unaffected by azure C, and anaerobic incubations are only mildly toxic (data not shown). The lethality observed in glucose minimal medium containing chloramphenicol is probably enhanced by the inability of E. coli B to induce superoxide dismutase under these conditions. The toxicity of azure C is relieved by the hydroxyl radical scavenger dimethylsulfoxide and the H202 scavenger catalase (Fig. 4). Furthermore, high intracellular levels of superoxide dismutase and catalase also relieve azure C toxicity. Almost complete protection is obtained by addition of dimethyl sulfoxide and exogenous Cu,Zn-SOD and catalase to E. coli B containing elevated intraceUular levels of superoxide dismutase and catalase (Fig. 4). Acknowledgments This work was supported by Public Health Service Research Grants AI-19695 and GM-07833, Grant C-900from the Robert A. Welch Foundation, and a grant from the American Heart Association Texas Affiliate.
29 H. Nikaido and E. Y. Rosenberg, J. Bacteriol. 153, 241 (1983). 3o j. S. Bellin, L. Lutwick, and B. Jonas, Arch. Biochem. Biophys. 132, 157 0969). 31 H. Nikaido, E. Y. Rosenberg, and J. Foulds, J. Bacteriol. 153, 232 (1983). 32 H. M. Hassan and I. Fridovich, J. Bacteriol. 141, 156 0980). 33 H. M. Hassan and I. Fridovich, J. Biol. Chem. 2,52, 7667 (1977).
