Vol. 276, No. 1, January,
pp. 139-145, 1990
Superoxide-Dependent and Superoxide-Independent Pathways for Reduction of Nitroblue Tetrazolium in Isolated Rat Cardiac Myocytes’ William
Departments of Pathology and Biological Chemistry, Broad and Vine Streets,wkhiladelph& Pennsylvan&
Received April 5,1989, and in revised form September
School of Medicine, 19102
Spectroscopic studies indicated that nitroblue tetrazolium (NBT) could be reduced to blue formazan by several distinct reactions in suspensions of isolated rat cardiac myocytes. Both NADPHand NADH-linked pathways for reduction of NBT were observed. NADPH-linked NBT reduction showed little activity in the absence of digitonin, but could be stimulated an average of 9.5-fold by digitonin permeabilization of the plasma membrane. NADH-linked NBT reduction occurred in the absence of digitonin, and could be increased an average of 3.5-fold by digitonin treatment. Analysis of the effects of cell viability on the extent of digitonin stimulation with these substrates suggested that the NADPHlinked reaction involved a cytosolic component, while the NADH-linked reaction involved an intracellular membrane enzyme system. With either NADPH or NADH, NBT reduction was completely inhibited by dicoumarol (100 PM). Dicoumarol-insensitive NBT reduction could subsequently be observed following the addition of 2 mM cyanide, a level of cyanide known to inhibit cytosolic superoxide dismutase. Cyanide-stimulated, dicoumarol-insensitive NBT reduction was augmented by the presence of either antimycin or doxorubicin, two agents which enhance superoxide formation by different mechanisms. The results indicate the existence of multiple pathways for both superoxide-independent and superoxide-dependent reduction of NBT. Dicoumarol-insensitive, cyanide-stimulated NBT reduction may be useful as a spectroscopic probe for inc) 1990Academic press, he. traCeh&Lr superoxide fOrmatiOn.
’ This work was supported by Research Grant HL-‘27929 from the National Heart, Lung and Blood Institute, and, in part, by Research Scientist Development Award AA-00087 from The National Institute on Alcohol Abuse and Alcoholism. ‘To whom correspondence should be addressed. 0003.9861/90 $3.00 Copyright 0 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.
Tetrazolium dyes, particularly nitroblue tetrazolium (NBT),3 have been used extensively as histologic staining reagents for various dehydrogenase enzymes (1, 2). Early studies established that the staining reaction was linked to diaphorase-type activities (l), meaning flavoproteins common to the oxidation of several substrates. Heart muscle was stained most intensely by NBT among several tissues tested (2). The pattern of stain deposition suggested an interaction with mitochondria (2). More recently, reduction of similar tetrazolium dyes by a wide variety of cells has been used as an assay for cell viability or metabolic activation under specific conditions (3). NBT has also been found to react with superoxide anion and has been used as a probe for this oxygen radical with several in vitro systems (4-6). Mediation of NBT reduction by superoxide, as well as reaction of superoxide with alternative probes such as cytochrome c or epinephrine, is commonly established through inhibition of the trapping reaction by exogenous superoxide dismutase (SOD) (6, 7). This approach works well with isolated enzymes or cells such as phagocytes which produce superoxide in an extracellular environment. Such a strategy would not be expected to be adequate for detection of superoxide in an intracellular environment, however, because most cells contain endogenous SOD. The high catalytic efficiency of SOD renders competition by a probe ineffective. We hypothesized that intracellular superoxide might be detectable by an appropriate probe if endogenous SOD was inhibited [see also (8) 3. CuZnSOD, the isozyme present in the cytosol of mammalian cells, is inhibited by copper-binding agents such as cyanide and diethyldithiocarbamate (DDC), while compounds such as azide are ineffective (9-12). In the present study, we have ” Abbreviations used: DDC, diethyldithiocarbamate; hydroxyethyl)-1-piperazine ethanesulfonic acid; NBT, razolium; SOD, superoxide dismutase.
Hepes, 4-(2nitroblue tet-
S. THAYER 2.5 z 2.0 s E 2 1.5 .E A ; 1.0 5
Digitonin bg/mg protein) FIG. 2. maximum (0.81 mg amounts addition shown.
Effect of digitonin concentration on NBT reduction. The rate of NBT reduction (squares) by rat cardiac myocytes protein/ml) was determined following addition of various of digitonin, as in Fig. IA. The lag time (circles) between of digitonin and attainment of the maximum rate is also
.,/ B/ t NBT
t Digitonin NBT TIME -
of NBT reduction by isolated heart cells. FIG. 1. Characteristics Rat cardiac myocytes (0.45 mg protein/ml) were incubated at 37°C in a Krebs-Ringer-Hepes medium at pH 7.4. NBT (10 PM), digitonin (10 pg/mg protein), and dicoumarol(lO0 FM) were added where indicated. (A) Requirement for digitonin permeabilization for rapid-phase NBT reduction; (B) effect of time of incubation with digitonin on rapidphase NBT reduction; (C) inhibition of rapid-phase NBT reduction by dicoumarol.
characterized the reaction of NBT in isolated cardiac myocytes and explored the feasibility of using NBT reduction as a probe for superoxide in cells treated with inhibitors of endogenous SOD. Our reason for undertaking this study was to develop a method for studying the mechanism by which the anticancer agent doxorubicin stimulates intracellular superoxide production. Considerable evidence has been amassed supporting such an occurrence in subcellular organelles, but characterization of this reaction in whole cells has been limited (13, 14). A preliminary report describing part of this work has appeared in abstract form (15). MATERIALS
Cardiac myocytes were isolated by collagenase perfusion of adult rat hearts from 300. to 500-g male Sprague-Dawley rats according to the
procedure described by Hohl et al. (16). Cells were resuspended in the low-K+ buffer with 2% bovine serum albumin as described (16) and maintained under 95% Op/5% CO:! at 37°C. For spectrophotometric experiments, cells were added to a Krebs-Ringer medium (16) containing 25 mM Hepes buffer at pH 7.4 at a final concentration of 0.42 mg cell protein/ml. Experiments were conducted within 2 h of cell preparation, during which time cell viability was typically 70-90%. Viability was estimated by light microscopy as rod-shaped cells excluding trypan blue (16) and by release of lactic dehydrogenase. Reduction of NBT was monitored by dual-wavelength spectroscopy at 560 - 750 nm using an Aminco DW-2A spectrophotometer. The dual-wavelength method was advantageous with cell suspensions because it permitted use of a single cuvette, thus eliminating variations in light scattering inherent with the use of separate sample and reference cuvettes. The extinction coefficient for NBT reduction measured at this wavelength pair was determined from calibration spectra to be about 85% of that at 560 nm (5) and corresponded to a value of 16.5 mMmL Cm -I. RESULTS
Characteristics of NBT reduction in cardiac myocytes. Suspensions of isolated rat heart cells showed little spontaneous reduction of NBT. After addition of digiTABLE Differential
Effect of Digitonin and NADH-Linked
Permeabilization NBT Reduction”
NBT reductionb Substrate NADPH NADH
1.08 +- 0.33 3.67 +- 1.93
11.0 & 3.25
9.69 k 3.16
Ratio (+/-)’ 9.52 14.12 3.52 + 1.59
’ Heart cells (approx 1 mg protein/ml) were treated with digitonin (15 pg/mg protein) in the presence of either NADPH or NADH (100 PM), as indicated. The rate of reduction of NBT (40 FM) was measured. The number of cell preparations tested is indicated in parentheses. * Mean f SD nmol NBT reduced/min/mg cell protein. ‘Mean t SD ratio of rates in the presence and absence of digitonin.
Cell Viability (%)
3. Differential effect of cell viability on NADPH-versus NADH-linked NBT reduction in the absence of digitonin. The initial rate of reduction of NBT (40 FM) following addition of either NADPH (100 pM, squares) or NADH (100 pM, circles) to rat cardiac myocytes (approximately 1 mg protein/ml) was determined in the absence of digitonin. Viable cells were estimated microscopically as rod-shaped cells which excluded trypan blue. Each viability point represents a separate cell preparation.
tonin at concentrations sufficient to permeabilize the plasma membrane (17), rapid reduction of NBT ensued (Fig. 1). NBT reduction exhibited a lag phase following digitonin addition and then proceeded along a sigmoidal time course (Fig. 1A). Increasing digitonin concentrations decreased the lag time between addition of digitonin and attainment of a maximum rate (Fig. 2). Adding digitonin prior to NBT resulted in a lesser extent of NBT reduction as indicated by the decreased steep portion of the absorbance record (Fig. 1B). Dicoumarol (100 PM) blocked subsequent NBT reduction in digitonin permeabilized cells (Fig. 1C). Addition of exogenous SOD to the cuvette did not alter the time course for NBT reduction under these conditions (not shown). In all cases, upon light microscopic examination, the insoluble blue formazan reduction product appeared inside the cells rather than in the extracellular medium. The sigmoidal time course observed for spontaneous NBT reduction upon permeabilization of the cells with digitonin suggested that an endogenous substrate was leaking out from the cells concomitant with entry of NBT. We found that NADH or NADPH addition could prolong the rapid phase of reduction of NBT. These substrates showed different relative responses to digitonin, however. NADPH supported little NBT reduction prior to digitonin addition, but prolonged the rapid phase reduction after digitonin. NADH gave a substantial rate of NBT reduction without addition of digitonin, but could be further stimulated by permeabilization (Table I). In cell preparations demonstrating 70% or greater viability, the rate of NADH-supported NBT reduction was stimulated an average of 3.5-fold by digitonin, whereas the rate of NADPH-supported reduction was stimulated an average of 9.5-fold upon permeabilization.
Comparison of cell preparations exhibiting various degrees of viability showed that the initial rate of NADH-supported NBT reduction in the absence of digitonin increased with decreasing viability (Fig. 3). Concomitantly, the degree of stimulation by digitonin declined in poorer cell preparations. On the other hand, the rate of NADPH-supported NBT reduction remained low in preparations showing low viability, although the degree of stimulation by digitonin likewise declined. With either NADPH or NADH as substrate, dicoumarol (loo-150 PM) completely inhibited rapid phase NBT reduction. Cyanide reactivation of NBT reduction. Under conditions as described above where dicoumarol caused complete inhibition of NBT reduction driven by either endogenous substrates, NADPH, or NADH, we observed that addition of KCN could partially restore NBT reduction (Fig. 4). Studies of the concentration dependence for the KCN effect showed that relatively high (millimolar) KCN levels were required to obtain NBT
0A560.75,, = 0.0 1 T
2 mM KCN
20 ~JM KCN
TIME FIG. 4. Effect of KCN on dicoumarol-insensitive NBT reduction in isolated heart cells. Rat cardiac myocytes (0.45 mg protein/ml) were incubated at 37°C in Krebs-Ringer-Hepes medium. Digitonin (22 fig/ mg protein), dicoumarol (100 KM), KCN or NaN,, and NBT (40 FM) were added where indicated. (A) 2 mM KCN, (B) 20 pM KCN, (C) 2 mM NaN,,
S. THAYER TABLE Effect
of Oxygen on Dicoumarol-Insensitive, KCN-Stimulated NBT Reduction” NBT reduction *
FIG. 5. KCN concentration dependence for stimulation of dicoumarol-insensitive NBT reduction in isolated heart cells. The effect of various concentrations of KCN on dicoumarol-insensitive NBT reduction was tested as in Fig. 4.
reduction (Figs. 4A, 4B, and 5). Azide was much less effective (Fig. 4C). By contrast, a lower level of KCN (0.1 mM) was sufficient to inhibit respiration completely in isolated cardiac myocytes (Fig. 6) or heart mitochondria. The KCN concentration dependence for stimulation of dicoumarol-insensitive NBT reduction was similar to that for inhibition of CuZnSOD (6, 9, lo), suggesting that inhibition of cytosolic SOD permitted NBT to trap endogenous superoxide. Model system studies with commercial CuZnSOD from bovine erythrocytes (Sigma Chemical Co.) verified the ability of KCN to inhibit this enzyme at similar millimolar concentrations, as has been noted previously (10). We likewise tested the effectiveness of DDC in place of KCN, since DDC is also recognized as an inhibitor of CuZnSOD (11). DDC was less effective at reactivation of dicoumarol-insensitive NBT reduction when compared to KCN in the cardiac
FIG. 6. Inhibition of respiration by KCN in isolated heart cells. The rate of oxygen uptake by isolated rat cardiac myocytes (0.41 mg cell protein/ml) in Krebs-Ringer-Hepes medium was measured polarographically with an oxygen electrode at 37°C. The medium contained an uncoupler, carbonyl cyanide m-chlorophenylhydrazone (1 FM), and various concentrations of KCN, as indicated. The 100% value corresponded to 86 natom O/min/mg protein.
Endogenous + NADPH + NADH
0.22 0.20 0.09
Anaerobic 0.17 0.05 0.00
Differenced 0.05 0.15 0.09
’ Heart cells (0.5 mgprotein/ml) were incubated with digitonin (20.5 (100 rg/mg protein), dicoumarol (150 PM), NBT (40 PM), NADPH FM), and NADH (100 PM), as indicated. * Values indicate the increase in the rate of NBT reduction (nmol/ min/mg cell protein) caused by the presence of KCN (8.5 mM) above the rate measured in the absence of KCN. ’ N,-bubbled medium was added to the cuvette and 0.5 ml of light silicone oil was layered on top of the solution. d Aerobic value minus the anaerobic value, which should be equivalent to the rate of superoxide formation.
myocyte system (not shown). Model system studies using commercial SOD showed, however, that DDC inhibition of SOD was time dependent, requiring as long as an hour to achieve full inhibition [see also (II)]. Thus, because cardiac myocytes could not withstand such a lengthy incubation with inhibitors, the effects of DDC on the myocyte system were not tested further. As an alternative approach to test whether the observed KCN-stimulated dicoumarol-insensitive NBT reduction involved superoxide formation, we studied the oxygen dependence of the reaction. We reasoned that superoxide formation would occur under aerobic, but not anaerobic, conditions. As shown in Table II, higher rates were indeed found under aerobic as compared to anaerobic conditions. However, some dicoumarol-insensitive, KCN-dependent NBT reduction was seen under anaerobic conditions. This may indicate the existence of additional superoxide-independent pathways for NBT reduction which can function in the absence of oxygen. Alternatively, the results may be indicative of incomplete removal of oxygen from the cuvette by N, bubbling. Effect of respiratory inhibitors on dicoumarol-insensiAs another approach to test tive NBT reduction. whether the KCN-dependent portion of dicoumarol-insensitive NBT reduction corresponded to superoxide formation, we examined the effects of respiratory inhibitors having known actions (Table III). Only antimycin was able to enhance the rate of NBT reduction beyond that produced by KCN alone under these conditions. It is noteworthy that rates of superoxide formation with submitochondrial particles are maximal in the presence of antimycin (18). Efect of dororubicin on NBT reduction. We also studied the effects of the anticancer agent doxorubicin (Adriamycin) on dicoumarol-insensitive NBT reduction. Studies with submitochondrial particles have
Effect of Respiratory Inhibitors on Dicoumarol-Insensitive NBT Reduction” Relative Inhibitor
14 38 21
100b 94 92
NBT reduction (rapid plus slow phases) showed a general increase with increasing doxorubicin concentration (Fig. 8). Similar results were obtained when either NADH or NADPH was present as substrate (Fig. 8). DISCUSSION
None Rotenone (2 GM) Malonate (5 mM) Antimycin (1.2 pg/mg protein)
a Heart cells (0.81 mg protein/ml) were treated with digitonin (12.3 Fg/mg protein), dicoumarol (100 PM), various inhibitors as indicated, either without or with KCN (5 mM). NBT (40 pM) was added last and the initial rate of NBT reduction was recorded. b The 100% value corresponded to 1.58 nmol NBT reduced/min/mg protein.
shown that doxorubicin can promote the formation of superoxide through a redox cycling interaction with the respiratory chain NADH dehydrogenase (19). Likewise, doxorubicin can stimulate superoxide formation by NADPH-cytochrome P450 reductase (20) and soluble flavoproteins such as xanthine oxidase (21). In the cardiac myocyte system, biphasic kinetics were observed for NBT reduction in the presence of doxorubicin (Fig. 7). An initial rapid reduction (“jump” or “burst” phase) was followed by a slower prolonged reduction phase. Comparison of recorder tracings obtained in the presence and absence of KCN using the dicoumarol-treated, digitonin permeabilized cells incubated with various concentrations of doxorubicin consistently showed a greater rate and extent of absorbance change with KCN present (Fig. 7). Both the KCN-dependent portion of the reaction (curves labeled “Difference” in Fig. 7) and total
Superoxide-independent reduction of NBT. Data presented in this paper indicate that there are several pathways for reduction of NBT in cardiac myocytes. These include NADH- and NADPH-linked reactions which can be inhibited by dicoumarol. The differential response between these substrates to digitonin permeabilization suggests a difference in subcellular location of the respective enzymes involved in NBT reduction. The most likely situation is that the NADPH-linked activity is associated with the cytosol, whereas the NADHlinked activity is associated with a membrane-bound intracellular organelle, probably mitochondria. This is suggested from comparison of the relative digitonin stimulations observed with cell preparations having different viability. Each myocyte preparation is a mixture of intact and leaky cells which have a damaged plasma membrane. Damaged cells would be expected to have lost their cytosolic components during the washing steps prior to final resuspension. Thus, if NADPH reacts with a cytosolic enzyme, rapid NBT reduction would be found only after the plasma membrane was disrupted; cells in the preparation that were leaky initially would lack the required cytosolic component and thus would be totally unreactive. Concomitantly, if NADH reacts with an intracellular membrane-bound enzyme, which is less likely to be lost during the washing procedures involved in the cell preparation, NADH would be expected to have access to its site of reaction in leaky cells
0.10 t +KCN
FIG. 7. Time course for NBT reduction in the presence of doxorubicin. (A) Rat cardiac myocytes (0.75 mg/ml) were incubated with digitonin (13.3 pg/mg protein), dicoumarol (150 PM), doxorubicin (400 PM), KCN (IO mM) where indicated, and NADPH (50 FM). Reduction of NBT (40 FM) was recorded. The curve labeled “Difference” was calculated by subtracting the “-KCN” trace from the “+KCN” trace. (B) A similar experiment except that concentrations were for myocytes, 0.55 mg/ml; for digitonin, 18.2 gg/mg protein; and for doxorubicin, 500 pM. NADH (50 PM), rather than NADPH, was present as substrate.
FIG. 8. Effect of doxorubicin concentration on NBT reduction. NBT reduction was measured as described in Fig. 7 with various concentrations of doxorubicin using either NADPH (left, squares) or NADH (right, circles) as substrate. Solid symbols indicate total NBT reduction during the initial 5 min following NBT addition (equivalent to “+KCN” traces in Fig. 7). Open symbols indicate the KCN-stimulated portion of total NBT reduction (equivalent to curves labeled “Difference” in Fig. 7).
prior to digitonin. Both intact and leaky cells would be accessible to NADH following digitonin treatment. The fact that dicoumarol inhibited both the NADPHand NADH-linked reactions suggests that these are “diaphorase” or dehydrogenase enzymes. In literature from the 1950s and 1960s the term diaphorase was used in a general way to refer to any enzyme catalyzing dye reduction (1). Subsequently, a specific enzyme, known as DTdiaphorase, was isolated from rat liver and characterized by Ernster (22). More recently, this same enzyme has been named quinone reductase (EC 22.214.171.124) (23, 24). It is a soluble flavoprotein which can utilize a number of quinone-containing dyes or drugs as subst,rate for twoelectron reduction with either NADPH or NADH. Such activity has been reported to be low in cardiac tissue (22). From our data, it would appear that quinone reductase (DT-diaphorase) is not one of the enzymes involved in tetrazolium dye reduction in rat heart myocytes. This statement is supported by the observation that considerably higher levels of dicoumarol (100 PM) are required to inhibit NBT reduction in the cardiac myocyte system than for the enzyme purified from rat liver (1 PM). In addition, tetrazolium dyes are not quinones. The precise identity of the particular enzymes responsible for NBT reduction remains to be determined. The data indicate, however, that the majority of NBT reduction in the heart occurs by non-superoxide-mediated routes. Superoxide-dependent reduction of NBT. A second set of pathways for NBT reduction is revealed upon addition of KCN at concentrations consistent with inhibition of cytosolic SOD. The fact that KCN-stimulated NBT reduction occurs in permeabilized cells indicates that it is associated with intracellular membrane systems. Effects of mitochondrial respiratory inhibitors, particularly that of antimycin, suggest that mitochon-
dria are likely a significant source for superoxide which can then be trapped by NBT in the cytosol. Mitochondrial superoxide formation is known to be maximal in the presence of antimycin (18). In addition, a site of superoxide formation located prior to the rotenone block in mitochondrial NADH dehydrogenase has been reported (19). The observation that doxorubicin enhanced the KCN-dependent portion of dicoumarol-insensitive NBT reduction, as well as total NBT reduction, also supports the interpretation that superoxide formation is being detected under these conditions. Superoxide formation catalyzed by doxorubicin has been demonstrated previously with heart submitochondrial particles (19, 25), intact mitochondria (25, 26), and heart “microsomal” fractions (26). The experimental observations reported in this study can be accommodated by the scheme shown in Fig. 9. In this scheme, superoxide-linked reduction of the NBT trapping reagent can be observed only after inhibition of endogenous SOD and after inhibition of the alternative dehydrogenase or nonspecific “diaphorase”-mediated pathways with dicoumarol. Under such conditions, the rate of NBT reduction would then be proportional to the rate of endogenous superoxide generation. The latter would be expected to be that occurring on the cytoplasmic side of the mitochondrial membrane. This is because MnSOD, present intramitochondrially, is not inhibited by CN- (9,lO). In some previous studies controversy has arisen concerning whether superoxide was generated by the presence of NBT itself, owing to formation of NBT radicals (8, 27-29). Such studies involved inclusion of phenazine methosulfate as a redox mediator between NADH and the tetrazolium dye (8,27). In such systems enzyme-catalyzed reduction of phenazine methosulfate
FIG. 9. Scheme for NBT reduction in isolated cardiac myocytes. Permeabilization of the plasma membrane of t,be myocytes by digitonin treatment is necessary for NBT reduction by allowing entry of NBT. Superoxide-independent NBT reduction occurs by nonspecific diaphorase (dehydrogenase)-mediated reactions linked to oxidation of NADPH (shown) or NADH (not shown). These pathways can be inhibited by dicoumarol (100 fiM). Superoxide-dependent NBT reduction can subsequently be observed following addition of KCN at a concentration suflicient to inhibit cytosolic CuZnSOD.
by NADH leads in turn to formation of NBT radical from a nonenzymatic reaction of the reduced phenazine. The NBT radical can subsequently generate superoxide by autoxidation (28, 29). In the present study, we have avoided using phenazine methosulfate or similar redox mediators to prevent such reactions. Under the conditions of our experiments, NBT is expected to trap, rather than form, superoxide radicals. Overall, the results of this study indicate that dicoumarol-insensitive, KCN-stimulated NBT reduction most likely does provide a measurement of intracellular superoxide formation. However, the complexity of the system, including the fact that digitonin permeabilization of the cells is necessary, limits the usefulness of this method. It can be noted that responses of other conventional probes for superoxide, including cytochrome c reduction and epinephrine co-oxidation, appeared similarly complex in preliminary experiments. Further understanding of the role of intracellular free radical reactions in problems of pathobiology would be aided by development of alternative spectroscopic probes for superoxide formation.
Doxorubicin stimulation of intracellular superoxide formation. Data presented in this paper indicate that doxorubicin can stimulate the dicoumarol-insensitive, KCN-stimulated portion of NBT reduction in a concentration-dependent manner. This would be consistent with enhancement of superoxide formation at increasing doxorubicin concentrations. However, the nature of the spectral reactions, particularly the biphasic kinetics of NBT reduction found in the presence of doxorubicin, is complex. It is likewise possible that the initial “burst” or “jump” phase of NBT reduction seen in the presence of doxorubicin may also arise from superoxide. Considerable evidence from both in vitro (13,19-21,25,26) and in viuo (14,30) studies now supports the hypothesis that free radical reactions subsequent to redox cycling by doxorubicin are indeed involved in the unique cardiotoxicity attributed to this drug. However, the pathogenic mechanism leading from free radical formation to cardiomyopathy has many aspects. The present results provide further support for the idea that doxorubicin can initiate free radical reactions directly within heart myocytes, although this may not be its exclusive mechanism of action.
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