670
ORGAN, TISSUE, AND CELL DAMAGE
[73]
[73] Assay for Free Radical Reductase Activity in Biological Tissue by Electron Spin Resonance Spectroscopy By JORGEN FUCHS, ROLF J. MEHLHORN, and LESTER PACKER
Introduction A variety of antioxidant defense systems have evolved in aerobic organisms to protect against oxidative stress) Reactive oxygen species include free radicals and electronically excited molecules like singlet oxygen. Both enzymatic and nonenzymatic mechanisms serve to intercept oxidants and repair damage. Free radicals can arbitrarily be divided into three classes: highly reactive radicals, e.g., hydroxyl, moderately reactive radicals, e.g., superoxide, and persistent radicals, e.g., ascorbyl. Kinetic considerations suggest that highly reactive radicals cannot be intercepted selectively by antioxidative systems. Intermediate and persistent free radicals, however, could be detoxified by specific protective mechanisms. Despite their low reactivity, weakly reactive radicals may pose a considerable threat to biomolecules, since they can react more selectively with targets at critical cell locations. If these radicals can be produced at high rates, e.g., like superoxide, then specific detoxifying systems like superoxide dismutase can confer effective protection. It is also plausible that persistent radicals, like those that arise from ascorbic acid, a-tocopherol, 2 and, perhaps, fl-carotene during reactions with reactive free radicals, may be reduced by specific enzymes that regenerate the original antioxidant. We have developed a simple assay for the free radical-reducing activity of tissue that is based on the one-electron reduction of nitroxide radicals. The nitroxides are relatively persistent radicals 3 whose concentration can easily be quantitated by electron spin resonance (ESR) spectroscopy. This assay had been applied to epidermis and skin homogenares, since the skin is a readily accessible tissue and a target organ of oxidative stress a which is subject to a variety of pathologic conditions that have been associated with free radical processes.
z H. Sies (ed.), "Oxidative S t r e s s , " Academic Press, New York, 1985. 2 p. McCay, Fed. Proc., Fed. Am. Soc. Exp. Biol. 45, 451 (1986).
3 E. G. Rozentsev(ed.), "Free NitroxylRadicals." Plenum,New York, 1970. 4y. Niwa, T. Kanoh, T. Sakane, H. Soh, S. Kawai, and Y. Miyachi,Life Sci. all, 912 (1987). METHODS IN ENZYMOLOGY,VOL. 186
Copyright © 1990by Academic Press, Inc, All rightsof reproductionin any form reserved.
[73]
E S R ASSAY FOR FREE RADICAL REDUCTASE ACTIVITY
671
Methods
Skin Preparation from Hairless Mice Female hairless mice from Jackson Laboratory (Bar Harbor, ME), 1012 weeks old, are sacrificed by CO2 inhalation followed by decapitation. This procedure avoids anesthetics which interfere with free radical metabolism. After a longitudinal cut through the back skin, the whole skin is manually separated from the body and washed blood-free with an isotonic buffer (130 mM sodium chloride, 5 mM glucose, 1 mM disodium EDTA, 10 mM sodium phosphate, pH 7.0). Adherent subcutis and fascia are removed by gently scraping the dermal side with tissue paper. The epidermal/dermal skin sheet is kept on glass wool pads (Type A/E, 47 ram, Millipore Corp., Bedford, MA). The pads are well saturated with isotonic buffer and ket in petri dishes on ice. Skin Homogenate. Skin homogenates are prepared by cutting skin from hairless mice (2 g) into small pieces that are homogenized with an Ultra-Turrax tissue blender (Tekmar Company, Cincinnati, OH) in an isotonic buffer (5-10 ml), as described for skin preparation, and subsequently with a Teflon pestle in a tight-fitting glass vessel. During homogenization the sample is continuously flushed with argon gas to prevent oxidation of reactive tissue components. The homogenate can be frozen and stored on liquid nitrogen. Soluble enzymes, like catalase, superoxide dismutase, glutathione peroxidase, and glutathione reductase can be assayed from the supernatant of the homogenate, after separating membranes and other tissue fragments by centrifugation in an Eppendorf centrifuge for 25 min.
Electron Paramagnetic Resonance Spectroscopy In the examples shown, first-derivative EPR spectra (100 kHz modulation frequency) are recorded at ambient temperature on a Bruker ER 200 D-SCR spectrometer (X-band). Modulation amplitude is 1.25 G, scan range I00 G, and microwave power I0 roW. Nitroxide concentrations are determined from the peak-to-peak height of the low-field line of the first derivative spectrum and calibrated according to a nitroxide standard. Nitroxide reduction in skin homogenate is measured in 75-ttl quartz glass capillaries. Skin biopsies are cut with a punch biopsy needle, care being taken to cut biopsies of similar sizes (surface area). Depending on the special characteristics of the EPR instrument and cavity used, skin biopsies 4 mm in diameter are the maximum size that allow for adequate cavity tuning. However, with some dehydration, experiments are feasible with 6 mm biopsies. Reduction of nitroxides in epidermis/dermis biopsies
672
ORGAN, TISSUE, AND CELL DAMAGE
[73]
is measured by placing a 4-mm biopsy in the lower end of a quartz glass tube (2.5 mm i.d.) and centering the tissue sample in the middle of the EPR cavity. Repetitive measurements of different samples result in a tuning error of less than 5% of the total signal observed. Chemicals. The uncharged nitroxide Tempol (2,2,6,6-tetramethyl-1piperidinoxy-4-ol) and the cationic nitroxides CAT-1 (2,2,6,6-tetramethyl1-piperidinoxy-4-trimethylammoniumbromide) and CAT-4 (2,2,6,6-tetramethyl-l-piperidinoxy-4-N,N-dimethyl-N-butylammonium bromide) are obtained from Molecular Probes (Junction City, OR). N-Ethylmaleimide (NEM), ascorbate oxidase (AO), and oxidized and reduced nicotinamide adenine dinucleotide diphosphate (NADP, NADPH) are purchased from Sigma (St. Louis, MO). 1,3-Bis[2-chloroethyl]- 1-nitrosourea (BCNU) was a gift from Dr. M. Smith, Berkeley, CA.
Analysis of Nitroxide Reduction in Skin Homogenates Reduction of piperidinoxy and dihydropyrrolinoxy spin probes can be used to discriminate between major reducing agents in biological tissue? Ascorbic acid, which is usually the most important reductant for nitroxides in biological tissues, is a potent reductant for piperidinoxyl radicals (six-membered rings), but has little effect on dihydropyrrolidinoxyl radicals (five-membered rings). In tissue homogenates, the influence of ascorbic acid in free radical reduction can also be studied by using AO and measuring the residual nitroxide-reducing activity. The involvement of thiol groups in free radical reduction is assessed by treatment of the homogenates with NEM or zinc. The residual nitroxide-reducing activity in skin homogenates after treatment with AO is NEM sensitive (Fig. 1). NEM-sensitive nitroxide reduction can be stimulated by NADPH and NADP, the latter being abolished by BCNU, a glutathione reductase inhibitor (Fig. 2). The involvement of thiol groups in nonascorbic acidmediated nitroxide scavenging is also demonstrated by a stimulation of reducing activity (data not shown) by mammalian thioredoxin,6 a protein that works as a disulfide reductant for other enzymes and low molecular weight compounds. Discrimination between enzymatic and nonenzymatic reduction can be achieved by heat treatment, exercising due care to consider the possible activation of transition metal-catalyzed reactions, which could occur when enzyme denaturation releases iron or copper. 5 S. Belkin, R. J. Mehlhorn, K. Hideg, O. Hankovsky, and L. Packer, Arch. Biochem. Biophys. 256, 232 (1987). 6 A. Holmgren, Annu. Reo. Biochem. 54, 237 (1985).
[73]
ESR ASSAY FOR FREE RADICAL REDUCTASEACTIVITY
60
50
=
control
•
AO NEM/AO
673
i ,o g so
2O
0
100
200
300
400
time [see] FIG. 1. Reduction of the membrane-permeable spin probe Tempol in skin homogenates and its inhibition by N-ethylmaleimide (NEM) and ascorbate oxidase (AO). Experimental conditions: reaction volume, 100/zl; 22°; 200 mg skin wet weight/ml homogenization medium; Tempol concentration, 100/zM; NEM final concentration, 25 mM; AO, 25 U/ml. Experiments were done in triplicate; mean values and standard deviation are shown.
250
le u A =
200
control NADPH NADP NADP/BCNU
8 i
150
=~ 8
loo
"7
50
~ o
0
I
0
100
200 tlme~]
300
400
FIG. 2. CAT-1 reduction in skin homogenates and its stimulation by NADP(H). Experimental conditions: reaction volume, 100/xl; 400 mg skin wet weight/ml homogenization medium; CAT-1 concentration, 200/zM (control) (CAT-I is a cationic membrane-permeable spin probe which partitions in the aqueous phase); NADPH final concentration, 10 raM; NADP, l0 mM; BCNU, 100/zM plus 10 mM NADP. Experiments were repeated at least four times; mean values and standard deviation are shown.
674
ORGAN, TISSUE, AND CELL DAMAGE •- - - e - - -
120
§•-100 g
----e---
[73] CAT-4 CAT4/heat CAT-4/Zn CAT-4/NEM
60 4o
g
.* 20 ¢J
0
0
I
I
I
100
200
300
400
time [see] FIG. 3. Nitroxide reduction at the epidermal surface of skin biopsies. The cationic membrane-binding nitroxide CAT-4, which partitions between the membrane surface and aqueous compartment, was applied on the epidermal side of a 4-ram skin biopsy (CAT-4, control). Biopsies were immersed in 70~ isotonic saline for 5 min and then exposed to CAT-4 (CAT-4/heat), 10 mM zinc sulfate (CAT-4/Zn), and 50 mM NEM (CAT-4/NEM) prior to nitroxide treatment. Representative experiments are shown.
Free Radical Reduction at Epidermal Surface of Skin Biopsies The epidermal surface of 4-mm skin biopsies is treated with 2/~1 of a 10 mM solution of nitroxide, incubated for 5 min, and washed for 15 sec with isotonic saline. Subsequently, the sample is immediately transferred to the EPR cavity and the nitroxide spectrum recorded. Nitroxide uptake into the epidermal membranes varies considerably and is roughly a function of the octanol-water partition coefficient. The epidermis can be pretreated with skin-permeable thiol group inhibitors, like NEM, treated with zinc sulfate, or heated briefly (70°, 5 min). A heat-, zinc-, and NEMsensitive free radical reducing component was identified in the epidermis with the membrane-binding cationic nitroxide CAT-4 (Fig. 3). The results confirm the finding of a "free radical reductase" at the surface of the epidermis7; the nature of the reductant, however, remains to be characterized. 8-1o 7 K. U. Schallreuter, M. R. Pittelkow, and J. M. Wood, J. Invest. Dermatol. 87, 728 (1986). 8 j. Fuchs, J. Invest. Dermatol. 91, 92 (1988). 9 K. U. SchaUreuter, M. P. Pittelkow, and J. M. Wood, J. Invest. Dermatol. 91, 92 (1988). 1oj. Fuchs, R. J. Mehlhorn, and L. Packer, J. Invest. Dermatol. submitted (1988).
ORGAN, TISSUE, AND CELL DAMAGE
[73]
[73] Assay for Free Radical Reductase Activity in Biological Tissue by Electron Spin Resonance Spectroscopy By JORGEN FUCHS, ROLF J. MEHLHORN, and LESTER PACKER
Introduction A variety of antioxidant defense systems have evolved in aerobic organisms to protect against oxidative stress) Reactive oxygen species include free radicals and electronically excited molecules like singlet oxygen. Both enzymatic and nonenzymatic mechanisms serve to intercept oxidants and repair damage. Free radicals can arbitrarily be divided into three classes: highly reactive radicals, e.g., hydroxyl, moderately reactive radicals, e.g., superoxide, and persistent radicals, e.g., ascorbyl. Kinetic considerations suggest that highly reactive radicals cannot be intercepted selectively by antioxidative systems. Intermediate and persistent free radicals, however, could be detoxified by specific protective mechanisms. Despite their low reactivity, weakly reactive radicals may pose a considerable threat to biomolecules, since they can react more selectively with targets at critical cell locations. If these radicals can be produced at high rates, e.g., like superoxide, then specific detoxifying systems like superoxide dismutase can confer effective protection. It is also plausible that persistent radicals, like those that arise from ascorbic acid, a-tocopherol, 2 and, perhaps, fl-carotene during reactions with reactive free radicals, may be reduced by specific enzymes that regenerate the original antioxidant. We have developed a simple assay for the free radical-reducing activity of tissue that is based on the one-electron reduction of nitroxide radicals. The nitroxides are relatively persistent radicals 3 whose concentration can easily be quantitated by electron spin resonance (ESR) spectroscopy. This assay had been applied to epidermis and skin homogenares, since the skin is a readily accessible tissue and a target organ of oxidative stress a which is subject to a variety of pathologic conditions that have been associated with free radical processes.
z H. Sies (ed.), "Oxidative S t r e s s , " Academic Press, New York, 1985. 2 p. McCay, Fed. Proc., Fed. Am. Soc. Exp. Biol. 45, 451 (1986).
3 E. G. Rozentsev(ed.), "Free NitroxylRadicals." Plenum,New York, 1970. 4y. Niwa, T. Kanoh, T. Sakane, H. Soh, S. Kawai, and Y. Miyachi,Life Sci. all, 912 (1987). METHODS IN ENZYMOLOGY,VOL. 186
Copyright © 1990by Academic Press, Inc, All rightsof reproductionin any form reserved.
[73]
E S R ASSAY FOR FREE RADICAL REDUCTASE ACTIVITY
671
Methods
Skin Preparation from Hairless Mice Female hairless mice from Jackson Laboratory (Bar Harbor, ME), 1012 weeks old, are sacrificed by CO2 inhalation followed by decapitation. This procedure avoids anesthetics which interfere with free radical metabolism. After a longitudinal cut through the back skin, the whole skin is manually separated from the body and washed blood-free with an isotonic buffer (130 mM sodium chloride, 5 mM glucose, 1 mM disodium EDTA, 10 mM sodium phosphate, pH 7.0). Adherent subcutis and fascia are removed by gently scraping the dermal side with tissue paper. The epidermal/dermal skin sheet is kept on glass wool pads (Type A/E, 47 ram, Millipore Corp., Bedford, MA). The pads are well saturated with isotonic buffer and ket in petri dishes on ice. Skin Homogenate. Skin homogenates are prepared by cutting skin from hairless mice (2 g) into small pieces that are homogenized with an Ultra-Turrax tissue blender (Tekmar Company, Cincinnati, OH) in an isotonic buffer (5-10 ml), as described for skin preparation, and subsequently with a Teflon pestle in a tight-fitting glass vessel. During homogenization the sample is continuously flushed with argon gas to prevent oxidation of reactive tissue components. The homogenate can be frozen and stored on liquid nitrogen. Soluble enzymes, like catalase, superoxide dismutase, glutathione peroxidase, and glutathione reductase can be assayed from the supernatant of the homogenate, after separating membranes and other tissue fragments by centrifugation in an Eppendorf centrifuge for 25 min.
Electron Paramagnetic Resonance Spectroscopy In the examples shown, first-derivative EPR spectra (100 kHz modulation frequency) are recorded at ambient temperature on a Bruker ER 200 D-SCR spectrometer (X-band). Modulation amplitude is 1.25 G, scan range I00 G, and microwave power I0 roW. Nitroxide concentrations are determined from the peak-to-peak height of the low-field line of the first derivative spectrum and calibrated according to a nitroxide standard. Nitroxide reduction in skin homogenate is measured in 75-ttl quartz glass capillaries. Skin biopsies are cut with a punch biopsy needle, care being taken to cut biopsies of similar sizes (surface area). Depending on the special characteristics of the EPR instrument and cavity used, skin biopsies 4 mm in diameter are the maximum size that allow for adequate cavity tuning. However, with some dehydration, experiments are feasible with 6 mm biopsies. Reduction of nitroxides in epidermis/dermis biopsies
672
ORGAN, TISSUE, AND CELL DAMAGE
[73]
is measured by placing a 4-mm biopsy in the lower end of a quartz glass tube (2.5 mm i.d.) and centering the tissue sample in the middle of the EPR cavity. Repetitive measurements of different samples result in a tuning error of less than 5% of the total signal observed. Chemicals. The uncharged nitroxide Tempol (2,2,6,6-tetramethyl-1piperidinoxy-4-ol) and the cationic nitroxides CAT-1 (2,2,6,6-tetramethyl1-piperidinoxy-4-trimethylammoniumbromide) and CAT-4 (2,2,6,6-tetramethyl-l-piperidinoxy-4-N,N-dimethyl-N-butylammonium bromide) are obtained from Molecular Probes (Junction City, OR). N-Ethylmaleimide (NEM), ascorbate oxidase (AO), and oxidized and reduced nicotinamide adenine dinucleotide diphosphate (NADP, NADPH) are purchased from Sigma (St. Louis, MO). 1,3-Bis[2-chloroethyl]- 1-nitrosourea (BCNU) was a gift from Dr. M. Smith, Berkeley, CA.
Analysis of Nitroxide Reduction in Skin Homogenates Reduction of piperidinoxy and dihydropyrrolinoxy spin probes can be used to discriminate between major reducing agents in biological tissue? Ascorbic acid, which is usually the most important reductant for nitroxides in biological tissues, is a potent reductant for piperidinoxyl radicals (six-membered rings), but has little effect on dihydropyrrolidinoxyl radicals (five-membered rings). In tissue homogenates, the influence of ascorbic acid in free radical reduction can also be studied by using AO and measuring the residual nitroxide-reducing activity. The involvement of thiol groups in free radical reduction is assessed by treatment of the homogenates with NEM or zinc. The residual nitroxide-reducing activity in skin homogenates after treatment with AO is NEM sensitive (Fig. 1). NEM-sensitive nitroxide reduction can be stimulated by NADPH and NADP, the latter being abolished by BCNU, a glutathione reductase inhibitor (Fig. 2). The involvement of thiol groups in nonascorbic acidmediated nitroxide scavenging is also demonstrated by a stimulation of reducing activity (data not shown) by mammalian thioredoxin,6 a protein that works as a disulfide reductant for other enzymes and low molecular weight compounds. Discrimination between enzymatic and nonenzymatic reduction can be achieved by heat treatment, exercising due care to consider the possible activation of transition metal-catalyzed reactions, which could occur when enzyme denaturation releases iron or copper. 5 S. Belkin, R. J. Mehlhorn, K. Hideg, O. Hankovsky, and L. Packer, Arch. Biochem. Biophys. 256, 232 (1987). 6 A. Holmgren, Annu. Reo. Biochem. 54, 237 (1985).
[73]
ESR ASSAY FOR FREE RADICAL REDUCTASEACTIVITY
60
50
=
control
•
AO NEM/AO
673
i ,o g so
2O
0
100
200
300
400
time [see] FIG. 1. Reduction of the membrane-permeable spin probe Tempol in skin homogenates and its inhibition by N-ethylmaleimide (NEM) and ascorbate oxidase (AO). Experimental conditions: reaction volume, 100/zl; 22°; 200 mg skin wet weight/ml homogenization medium; Tempol concentration, 100/zM; NEM final concentration, 25 mM; AO, 25 U/ml. Experiments were done in triplicate; mean values and standard deviation are shown.
250
le u A =
200
control NADPH NADP NADP/BCNU
8 i
150
=~ 8
loo
"7
50
~ o
0
I
0
100
200 tlme~]
300
400
FIG. 2. CAT-1 reduction in skin homogenates and its stimulation by NADP(H). Experimental conditions: reaction volume, 100/xl; 400 mg skin wet weight/ml homogenization medium; CAT-1 concentration, 200/zM (control) (CAT-I is a cationic membrane-permeable spin probe which partitions in the aqueous phase); NADPH final concentration, 10 raM; NADP, l0 mM; BCNU, 100/zM plus 10 mM NADP. Experiments were repeated at least four times; mean values and standard deviation are shown.
674
ORGAN, TISSUE, AND CELL DAMAGE •- - - e - - -
120
§•-100 g
----e---
[73] CAT-4 CAT4/heat CAT-4/Zn CAT-4/NEM
60 4o
g
.* 20 ¢J
0
0
I
I
I
100
200
300
400
time [see] FIG. 3. Nitroxide reduction at the epidermal surface of skin biopsies. The cationic membrane-binding nitroxide CAT-4, which partitions between the membrane surface and aqueous compartment, was applied on the epidermal side of a 4-ram skin biopsy (CAT-4, control). Biopsies were immersed in 70~ isotonic saline for 5 min and then exposed to CAT-4 (CAT-4/heat), 10 mM zinc sulfate (CAT-4/Zn), and 50 mM NEM (CAT-4/NEM) prior to nitroxide treatment. Representative experiments are shown.
Free Radical Reduction at Epidermal Surface of Skin Biopsies The epidermal surface of 4-mm skin biopsies is treated with 2/~1 of a 10 mM solution of nitroxide, incubated for 5 min, and washed for 15 sec with isotonic saline. Subsequently, the sample is immediately transferred to the EPR cavity and the nitroxide spectrum recorded. Nitroxide uptake into the epidermal membranes varies considerably and is roughly a function of the octanol-water partition coefficient. The epidermis can be pretreated with skin-permeable thiol group inhibitors, like NEM, treated with zinc sulfate, or heated briefly (70°, 5 min). A heat-, zinc-, and NEMsensitive free radical reducing component was identified in the epidermis with the membrane-binding cationic nitroxide CAT-4 (Fig. 3). The results confirm the finding of a "free radical reductase" at the surface of the epidermis7; the nature of the reductant, however, remains to be characterized. 8-1o 7 K. U. Schallreuter, M. R. Pittelkow, and J. M. Wood, J. Invest. Dermatol. 87, 728 (1986). 8 j. Fuchs, J. Invest. Dermatol. 91, 92 (1988). 9 K. U. SchaUreuter, M. P. Pittelkow, and J. M. Wood, J. Invest. Dermatol. 91, 92 (1988). 1oj. Fuchs, R. J. Mehlhorn, and L. Packer, J. Invest. Dermatol. submitted (1988).
