ARCHIVES
OF BIOCHEMISTRY
Vol. 299, No. 1, November
AND
BIOPHYSICS
15, pp. 30-37,1992
Superoxide Formed from Cigarette Smoke Impairs Polymorphonuclear Leukocyte Active Oxygen Generation Activity Masahiko
Tsuchiya,’
David
F. T. Thompson,
Yuichiro
J. Suzuki,
Carroll
E. Cross,*
and Lester
Packer
Department of Molecular and Cell Biology, 251 LSA, University of California, Berkeley, California 94720; and *Department of Medicine and Physiology, University of California, Davis, California 95616
Received April 1, 1992, and in revised form July 21, 1992
Reactive free radicals contained in cigarette smoke (CS) and compromised phagocytic antimicrobial activities including those of polymorphonuclear leukocytes (PMNs) have been implicated in the pathogenesis of severe CS-related pulmonary disorders. In CS-exposed buffer solutions, 0, was the predominant generated reactive oxygen species, as demonstrated by lucigenin-amplified chemiluminescence and electron spin resonance (ESR) spin-trapping with 5,5-dimethyl-1-pyrroline Noxide (DMPO). When PMNs were incubated in this buffer, phorbol 12-myristate 13-acetate (PMA)-stimulated active oxygen production and coupled O2 consumption were strongly impaired without appreciably affecting PMN viability (1-min exposure inhibited active oxygen production by 75%). Superoxide dismutase (SOD) totally protected and an iron chelator, diethylenetriaminepentaacetic acid (DETAPAC), also protected the CS-exposed PMNs, suggesting that generated 0; was an initiating factor in the impairment and OH - generation was a subsequent injurious factor. Pretreatment of PMNs with antioxidants such as a-tocopherol and dihydrolipoic acid (DHLA) was partially protective. The results suggest that (i) 0; is probably generated in the upper and lower respiratory tract lining fluid when they come in contact with CS; (ii) such generated 0; can primarily impair PMN capabilities to generate reactive oxygen species; and (iii) since these effects may contribute to the pathogenesis of CS-related lung diseases, prior supplementation with antioxidants such as a-tocopherol or DHLA might be successful in preventing these deleterious effects. kc) 1992 Academic Press, Inc.
Cigarette smoking is associated with pulmonary disorders including predisposition for respiratory infections, 1 To whom correspondence
should be addressed.
pulmonary emphysema, and cancer (l-4). Although the exact mechanisms by which smoking produces these effects are incompletely understood, it has been hypothesized that the free radicals contained in or generated by CS2 may be among the potential causative factors (4, 5). The free radical species contained in CS have been extensively studied by Pryor, Church, and their coauthors (6-10). They reported various kinds of free radicals derived from CS itself, which are possibly toxic. To estimate the toxicity of these radicals in uiuo, however, radical species which are generated in CS-exposed aqueous solution should be also considered, because inspired CS first interacts with aqueous surfaces of the upper and lower respiratory tract lining fluid. OH * generation from aqueous extract of tar (11) and H202 generation in the CS-bubbled aqueous solution (12) have already been reported by Pryor and Nakayama, respectively. Pryor et al. provide evidence that 0; was generated as an intermediate in this reaction (11, 13-14). In addition, it has been demonstrated that these radicals induce single-strand breaks of DNA (1316). It was also reported by Cross et al. that the interaction of CS with plasma induced lipid peroxidation (17). Alveolar macrophages and PMNs are thought to play an essential role in antimicrobial defense of the lung. Once coming across noxious agents, these cells produce a flood of bactericidal reactive oxygen species, including O,, OH -, and HOCl/OCl-, a phenomenon known as the NADPH oxidase-mediated respiratory burst (18, 19). Several studies have suggested that smoking causes qualitative and quantitative alterations in these inflammatory ’ Abbreviations used: CS, cigarette smoke; DETAPAC, diethylenetriaminepentaacetic acid; DHLA, dihydrolipoic acid, DMPO, 5,5-Dimethyl-1-pyrroline N-oxide; ESR, electron spin resonance; hfc, hyperfine coupling constant; KRP, Krebs-Ringer-phosphate; lucigenin, lO,lO’-dimethyl his-9,9’-biacridinium dinitrate; luminol, 5-amino-2,3-dihydro-1,4phthalazinedione; PMA, phorbol-12-myristate-13-acetate; PMNs, polymorphonuclear leukocytes; SOD, superoxide dismutase.
30
000%9861/92 All
$5.00
Copyright 8 1992 by Academic Press, Inc. rights of reproduction in any form reserved.
SUPEROXIDE
FROM
CIGARETTE
phagocytic cells (20-26), which possibly increase the susceptibility to infection or damages airway alveolar and/ or lining fluid components involved in respiratory tract defense mechanisms. This may be an important process in the development of several CS-related diseases. Thus, the effects of CS on the functions of these phagocytes are of importance. Reduction of proteolytic activity (27), inhibition of chemotaxis (28), decrease in phagocytic and bactericidal activity (29, 30), and desensitization to chemotactic peptide (31) of macrophages and PMNs have been reported as in vitro primary effects of CS. However, to our knowledge, few studies have been focused on the direct effect of CS on the generation of antimicrobial (and potentially injurious) reactive oxygen species from these inflammatory and phagocytic cells. Therefore, the goal of the present research was to study the radical generation in CS-exposed aqueous solutions, and to determine whether CS exposure affects the respiratory burst of PMNs and whether CS-derived radicals play a role in the process. MATERIALS
AND
METHODS
Chemicals DMPO was purchased from Daiichi Pure Chemicals Co., Ltd. (Tokyo). Lucigenin, luminol, PMA, SOD (from bovine erythrocytes), and catalase (from bovine liver) were from Sigma Chemical Co. (St. Louis, MO). Xanthine oxidase from cow’s milk was from Boehringer Mannheim Biochemicals (Indianapolis, IN). The University of Kentucky (UK) 2RI standard cigarettes containing 23 mg tar and 2.2 mg nicotine per cigarette (according to the U.S. Federal Tobacco Council) were used. a-Tocopherol was a gift from Henkel Corp. (LaGrange, IL), DHLA was from Asta Medica (Frankfurt), and p-carotene was from Roche Vitamins and Fine Chemicals. (Nutley, NJ). Other reagents were commercial products of analytical grade.
Measurement Solution
of Radical Generation from CS in Aqueous
Chem&minescence assay. Unfiltered main stream CS from the standard cigarette was bubbled through a 20 mM phosphate buffer (pH (32, 33) for 7.4) containing 100 mM lucigenin and 0.1 mM DETAPAC 2 min at a flow rate of 50 ml/min using the method of Pryor et al. (7) (Fig. 1). After the termination of bubbling, lucigenin-sensitive chemiluminescence of the buffer was recorded with a luminometer (LKB Wallac 1250, Finland). ESR spin trapping technique. Radical generation in the aqueous soluticn was also measured by the ESR spin trapping technique using the cyclic nitrone spin trap, DMPO. A DMPO solution (500 mM) with 0.5 mM DETAPAC was bubbled with the main stream CS for 5 min at a flow rate of 50 ml/min, as shown in Fig. 1. The solution was transferred to a flat quartz ESR cuvette (0.3.mm-thick flat cell, LABOTEC, Japan), which was placed in the cavity of the ESR spectrometer (IBM ER2OOD. SRC). Recordings of the spectra were made at modulation amplitude 1.25 G, modulation frequency 100 kHz, microwave power 9.6 mW, time constant 0.5 s, receiver gain 1 X 106, and sweep time 30 G/min. The hfc were measured directly from the scan field using MnO as an external standard. 0, spin adduct. The usual spectra of a 0; spin adduct of DMPO were recorded by the reaction of 100 mu/ml xanthine oxidase with 5 mM xanthine in a 30 mM phosphate buffer (pH 7.4), containing 100 mM DMPO and 0.1 mM DETAPAC.
SMOKE
IMPAIRS
FIG. 1.
31
NEUTROPHILS
System for exposure of CS to samples.
Measurement of PMNs Respiratory Burst and Effect of CS Exposure Preparation of PMNs. PMNs were isolated at 4°C from the peritoneal cavity of male Sprague-Dawley rats (250-300 g) 12 h after the intraperitoneal injection of 2% neutrose (2% neutralized casein sodium in norma! saline (pH 7.4)) as previously described (32,33). The isolated PMNs were suspended in KRP buffer, consisting of 154 mM NaCl, 6 mM KCl, 1 mM MgCl,, and 10 mM sodium phosphate buffer (pH 7.4). All measurements of PMNs were finished within 3 h after the start of the isolation. Measurementof active oxygen production. Production of reactive oxygen species from PMNs was measured by chemiluminescence and ESR spin trapping techniques. PMNs (2 X lo6 cells/ml) were incubated in a KRP buffer with 5 mM glucose, 500 pM calcium, and 100 pM lucigenin (or luminol) (for the chemiluminescence assay) or 50 mM DMPO (for the ESR spin trapping assay) and stimulated by the addition of 10 nM PMA. The chemiluminescence assays were performed using a luminometer, whereas the spin adduct of DMPO was measured with an ESR spectrometer using the same settings described above. Measurement of 0, consumption. 0, consumption coupled with the respiratory burst was measured polarographically with a Clark-type oxygen electrode. PMNs (1 X lo7 cells/ml) were incubated in KRP buffer with 10 mM glucose and 1 mM calcium and stimulated by 100 nM PMA. Measurement of cell uiability. PMN suspension was mixed with trypan blue (0.02% in final concentration) (36, 37). Since unviable cells could not exclude dye, the percentage of cells stained with dye is a measure of viability. Effect of CS on PMNs. PMNs in a KRP buffer were bubbled with main stream CS at a flow rate of 50 ml/min for 1 min as shown in Fig. 1; then active oxygen generation (chemiluminescence and ESR measurement), 0, consumption, and viability of PMNs were measured. In the case of chemiluminescence and ESR measurements, lucigenin (or luminol) or DMPO was added to the PMN suspension after the termination of exposure, which served to minimize or eliminate CS-derived free radical signals from PMN-derived active oxygen signals. Effect of 0; generated by ranthine and xanthine oridase on PMNs. After 2-min exposure to 0; generated by the reaction of 10 mu/ml xanthine oxidase with 50 FM xanthine, PMNs (2 X lo6 cell/ml) were stimulated by PMA, and lucigenin-amplified chemiluminescence was measured.
Protective Effect of Antioxidants
on CS Exposure
PMNs were preincubated for 5 min with various antioxidants (LYtocopherol, p-carotene, and DHLA) individually in order to allow incorporation of the antioxidant into the PMNs, and were washed with
32
TSUCHIYA
KRP buffer two times (38). These pretreated PMNs in a KRP buffer were bubbled by main stream CS at flow rate of 50 ml/mm for 1 min as shown in Fig. 1, and then their active oxygen generation activity induced by PMA was measured by lucigenin chemiluminescence and ESR spin trapping.
ET AL.
(A) MODEL SYSTEM XANTHINE +XANTHINE OXIDASE
Temperature and Oxygen Control All reactions were carried out at 37°C. Chemiluminescence and 0, consumption were measured using a thermostated cuvette equipped with a mixing device which maintained relatively homogeneous PMN suspension and supplied 0, to the PMNs from the atmosphere during the measurement. ESR spectra were recorded in a cell maintained thermostatically by nitrogen gas flow.
(8) SMOKE AQUEOUS
EXPOSED SOLUTION
Data Processing All experiments were repeated three to five times, and data are expressed as means f SD. The statistical significance of difference between two mean values was tested by Student’s t test.
RESULTS
Radical Generation from CS in Aqueous Solution CS exposure of phosphate buffer produced lucigenindependent chemiluminescence which is known to be highly sensitive to the 0; (39) (Fig. 2). The intensity of chemiluminescence gradually decreased after the termination of exposure. This chemiluminescence was totally eliminated by the addition of 100 units/ml SOD, indicating that 0; generation occurred in the CS-exposed buffer. The presence of 0; was confirmed by ESR spin trapping with DMPO. A typical DMPO-OOH (0, spin adduct of DMPO) adduct spectrum was generated by the xan-
I (C) pretreated with (D) AIR EXPOSED I
I
I
I SOD 01
I
T
(Cl EXPOSURE + SOD
FIG. 3. ESR spectra of spin adducts generated by CS exposure of aqueous solution in the presence of DMPO. Reaction mixture (300 ~1, 37°C) contained 0.5 mM DETAPAC in 500 mM DMPO solution before CS exposure. (A) Standard spectra of the 0; spin adduct of DMPO generated by 100 munits/ml xanthine oxidase plus 5 mM xanthine in 30 mM phosphate buffer (pH 7.4) containing 50 mM DMPO and 0.1 mM DETAPAC. (B) DMPO spin adduct spectra of 5-min CS-bubbled solution in the absence of SOD. (C) DMPO spin adduct spectra of 5-min CS-bubbled solution in the presence of 100 units/ml SOD at the beginning of CS exposure.
thine and xanthine oxidase system, and an almost identical spectrum (aN, 14.3 G; a;, 11.5 G; a:, 1.3 G) was observed in the CS-exposed DMPO solution in the company of a small amount of DMPO-OH (OH. spin adduct of DMPO: a,“, 14.8 G; a,!j, 14.8 G) (Fig. 3). The signal was not detected in the presence of 100 units/ml SOD or when the DMPO solution was bubbled with air in place of CS. Effect of CS Exposure on Reactive Oxygen Species Production by PMNs
I
6 4 2 INATION CFEQTSBE TIME IN MINUTES
SMOKE
8
FIG. 2. Lucigenin-dependent chemiluminescence generated in CSbubbled buffer. Reaction mixture (2 ml, 37’C) contained 100 mM lucigenin and 0.1 mM DETAPAC in 20 mM phosphate buffer (pH 7.4) before CS exposure. (A) 2-min CS-bubbled buffer. (B) 2-min CS-bubbled buffer + 100 units/ml SOD as indicated. (C) 2.min CS-bubbled buffer in the presence of 100 units/ml SOD at the beginning of CS exposure. (D) Control (2.min air-bubbled buffer).
After treatment with PMA, PMNs produce various reactive oxygen species, which can be monitored by either lucigenin or luminol-sensitive chemiluminescence (40). The chemiluminescence of both lucigenin and luminol was equally inhibited by exposure to CS in an exposureduration-dependent manner (Figs. 4 and 5), being almost totally inhibited by a 5-min CS exposure. The O2 concentration in PMN suspension in control and CS exposure was maintained at about 220 PM with a variation of 20 PM during measurement. Effect of CS Exposure on O2 Consumption
of PMNs
The respiratory burst of the PMNs is accompanied by a remarkable O2 consumption (41) (Fig. 6), which is an another reliable index of the PMN active oxygen gener-
SUPEROXIDE
6
n
WITH LUCIGENIN
A
WITH LUMINOL
E
8
FROM
CIGARETTE
60
B
SMOKE
IMPAIRS
erated, did not respond fully to the PMA stimulation (Fig. 7). The magnitude of the chemiluminescence response was less than half of that of PMNs which were not exposed to 0;. In control the presence of either 20 PM xanthine or 10 PM uric acid, the final product of xanthine oxidation, did not affect chemiluminescence generation by PMNs, and the inhibitory effect by 0, generation in the suspension was not affected in the presence of 8800 units/ml catalase, which eliminated another final product, Hz02, of xanthine oxidation. The O2 concentration in the PMN suspension was maintained at about 220 PM with a variation of 20 PM during measurement. Effect of Antioxidants
SMOKE EXPOSURE
TIME (SEC)
FIG. 4. Time dependent effect of CS exposure on PMNs respiratory burst measured by chemiluminescence. The reaction mixture (2 ml) contained 2 X 10s cells/ml PMNs, 154 mM NaCl, 6 mM KCl, 1 mM MgCl,, 5 mM glucose, and 500 FM CaCl, in 10 mM sodium phosphate buffer (pH 7.4) at 37°C. PMNs were stimulated by 10 nM PMA and their responses were estimated by measuring the area of generated chemiluminescence signal for 8 min. Closed square, chemiluminescence amplified by 100 pM lucigenin. Open triangle, chemiluminescence amplified by 100 FM luminol.
ation activity. Exposure of PMN suspension to CS reduced O2 concentration in the suspension by less than 10% of control and this reduction immediately recovered during the process for transfer of sample from CS exposure apparatus to oxygen electrode. The rate of O2 consumption in PMN activation was reduced by CS exposure, which agrees well with chemiluminescence results that CS impaired the respiratory burst. The presence of 100 units/ml SOD in the buffer markedly protected PMNs against CS exposure. SOD alone decreased the O2 consumption rate only slightly, possibly indicating enhanced dismutase reaction by exogenous SOD. The iron chelator, 10 PM DETAPAC (32, 33), which had no effect of PMN O2 consumption, also protected PMNs from the CS-induced inhibition.
33
NEUTROPHILS
on the CS Exposure
Based on the above results, it is expected that antioxidants might protect PMNs against smoke exposure. To estimate this protective effect, we determined how well the respiratory burst was maintained after CS exposure. The degree of effectiveness varied according to the antioxidant incorporated (Table II). In particular, cr-tocopherol-treated PMNs showed a good resistance to CS exposure (Fig. 5). Pretreatment of PMNs with the concentrations of antioxidants used did not affect active oxygen production of PMNs (data not shown). The protective effect of ol-tocopherol was also confirmed by spin trapping. The DMPO-OOH (DMPO-superoxide) signal followed by small DMPO-OH (DMPO-hydroxy radical) was observed in PMA-stimulated intact PMN suspension (42) (Fig. 8). CS exposure reduced these signals in untreated PMNs, but was only slightly affected in (Ytocopherol-pretreated PMNs. For measurement of the
Effect of CS Exposure on Cell Viability The percentage of PMNs stained with trypan blue did not increase significantly after 5 min exposure to CS at the highest dose used in any of our experiments (Table I). 6
Effect of Enzyme-Generated
0; on PMNs
To confirm toxicity of 0; generated in PMN suspension, 05 was enzymatically generated in the suspension under the same conditions as those of CS exposure. PMNs, which were previously incubated for 2 min in the buffer, where small amounts of 0; comparable to those generated in CS-exposed buffer were enzymatically gen-
TIME IN MINUTES FIG. 5. Effect of CS exposure on active oxygen generation by PMAstimulated PMNs. Active oxygen generation was monitored with lucigenin-sensitive chemiluminescence. PMNs were stimulated by 10 nM PMA. Conditions as described in the legend to Fig. 4 in the presence of 100 pM lucigenin. (A) Control (air exposure). (B) 1-min CS exposure to PMNs pretreated by 2.5 ~.LM a-tocopherol. (C) l-min CS exposure to PMNs.
34
TSUCHIYA
ET AL.
XANTHINE
0
(D) SMOKE EXPOSURE
/
8
4
12
16
0
TIME IN MINUTES I 0
f
1
I
4
a
12
I 16
20
TIME IN MINUTES FIG. 6. Effect of CS exposure on 0, consumption by PMNs coupled with active oxygen generation. Conditions as described in the legend to Fig. 4 except for concentration of PMN (1 X lo7 cells/ml) CaCl, (1 mM), glucose (10 mM) and PMA (100 nM). (A) Control (air exposure). (B) Air exposure in the presence of 10 PM DETAPAC at the beginning of exposure. (C) Air exposure in the presence of 100 units/ml SOD at the beginning of exposure. (D) 1.5.min CS exposure in the presence of 100 units/ml SOD at the beginning of exposure. (E) 1.5.min CS exposure in the presence of 10 HIM DETAPAC at the beginning of exposure. (F) 1.5-min CS exposure.
respiratory burst with ESR, DMPO was added to a PMN suspension after the termination of CS exposure, since this method of procedure allows the elimination or minimization of any contamination by CS-produced ESR signals. As a result, no ESR signal was observed in unstimulated CS-exposed PMN suspension, and therefore the signal recorded after PMN stimulation is considered to specifically represent reactive oxygen species generated by PMNs.
FIG. 7. by PMNs. of 100 pM xanthine nM PMA. enzymatic
Effect of enzyme-produced 0; on active oxygen generation Conditions as described in the legend to Fig. 3 in the presence lucigenin. 0; was generated by the reaction of 10 mu/ml oxidase with 50 pM xanthine. PMNs were stimulated by 10 (A) Control (no exposure to 0;). (B) Exposure to 0; generated reaction.
DISCUSSION
CS-related respiratory tract diseases are generally recognized to represent the combined effects of direct CS injury to the cellular component of respiratory tract and secondary inflammatory immune and infectious process related to the primary CS damage. Presumably, both CS and phagocytic reactive oxygen species play a role in this process. The role of CS oxidant is complex because of the multitude of free radical species known to be present in CS (7, 9), whereas the role of phagocytic oxidant is theo-
TABLE Effect
of Antioxidants of PMNs
Treatment TABLE Effect
of CS on Tryptan
II
on the Respiratory Exposed to CS
Burst
Chemiluminescence intensity (arbitrary unit)
I
Blue Dye Exclusion
in PMNs
Treatment
Cells strained with trypan hlue (percentage)
Control (air exposure) CS exposure (5 min)
2.0 f 1.4 5.9 k 2.6
Note. PMN suspension (containing 2 X 10” cells/ml PMNs, 154 mM NaCI, 6 mM KCl, 1 mM MgCl,, 5 mM glucose, and 500 FM CaCl* in 10 mM sodium phosphate buffer (pH 7.4, at 37°C) was bubbled by main stream of CS at a flow rate of 50 ml/min for 5 min as in Fig. 1, and diluted with trypan blue (0.02% in final concentration), and then the number of PMNs stained with dye was counted. The percentage was recorded by at least eight separate countings.
Control (air exposure) CS exposure + n-Tocopherol t DHLA /j-Carotene
100.0 20.0 t 0.4 47.0 i 5.4* 47.3 + 4.3-3
31.8 * 2.1*
Note. PMNs were preincubated for 5 min with 2.5 /rM of the antioxidant indicated, and exposed to main stream of CS at a flow rate of 50 ml/min for 1 min as in Fig. 1, and then stimulated with 10 nM PMA at 37°C. The respiratory burst of PMNs was monitored by measuring lucigenin-amplified chemiluminescence. Control measurements were performed with PMNs preincubated with solvent alone. The reaction mixture contained 2 X lo6 cells/ml PMNs, 154 mM NaCl, 6 mM KCl, 1 mM MgCl,, 5 mM glucose, 500 pM CaClz, and 100 fiM lucigenin in 10 mM sodium phosphate buff&r (pH 7.4). Each response of PMNs was estimated by measuring the area of chemiluminescence signal for 8 min. * Significantly different from CS exposure at P < 0.05.
SUPEROXIDE
FROM
CIGARETTE
(A) CONTROL
(B) SMOKE
/
EXPOSURE
ICI SMOKE
EXPOSURE
a-TOCOPHEROC TREATED PMNs
FIG. 8. Protective effect of n-tocopherol on CS-exposed PMNs. PMNs were stimulated by 10 nM PMA, and generated active oxygen was monitored by DMPO (50 mM) spin adduct formation 8 min after the stimulation. Conditions as described in the legend to Fig. 4. (A) Control (air exposure). (B) l-min CS exposure to PMNs. (C) I-min CS exposure to PMNs pretreated by 2.5 pM cu-tocopherol.
retically both adventitious (antimicrobial activity) and injurious (capable of oxidatively injuring neighboring cells including phagocytes). The present studies detailed some of the interactions of these two potent oxygen radical generating systems believed to be relevant when both are present in respiratory tract lining fluids; we focused especially on the known generation of 0, by both CS and PMNs and the possible consequences of their interaction. 0; is not only toxic itself (43, 44), but it also would lead to the generation of additional potentially and highly toxic metabolites such as H,O,, OH *, and ‘0, (45-47). If such 0, is formed at the surfaces of the upper and lower respiratory tract lining fluid when it comes in contact with CS, surrounding airway and lung parenchymal (alveolar) components, which form a mechanical barrier against microorganisms, may be seriously damaged. Thus damage may increase both airways and lung parenchymal susceptibility to infection. PMNs are an essential component of antimicrobial defenses of the lung. There have been several reports that there is a significant increase in the number of PMNs and/or the oxygen-derived free radicals from PMNs in the plasma and alveolar lavage fluid of cigarette smokers or CS-exposed animals (20, 22-24). The observed effect is interpreted as an activation effect of CS on PMNs, which conceivably leads to oxidative damage to airway or to alveoli. However, the “influx” and “activation” of PMNs into the lung may he due to the effect of release of chemical mediators secondary to tissue damage induced by CS, and the localized and primary effect of CS on phagocytes might then be masked. According to the tox-
SMOKE
IMPAIRS
NEUTROPHILS
35
icity of CS (30), PMNs present in the respiratory tract lining fluids which are directly exposed to CS might themselves be injured. It has been reported that CS directly inhibited PMN chemotaxis (28, 31), phagocytosis (29), and proteolytic (27) and bactericidal (30) activity in vitro. The regional failure of these inflammatory cells in CS-threatened airways may be more important in the development of CS-related lung disease. It has been shown by Pryor et al. that tar-derived radicals in CS had a capacity for producing 0, in aqueous solution and that ESR spin adduct signals from cigarette tar is attenuated by the SOD (9, 11, 13, 14). Nakayama et al. have also shown that Hz02 is formed when CS is bubbled through water, suggesting that the 0; is an intermediate (12). In t,he present study with chemiluminescence and ESR, it is directly demonstrated that 0; was generated in the CS-exposed aqueous solution, which confirmed Pryor’s previous findings (9, 11, 13, 14). A brief exposure of PMNs to CS inhibited their reactive oxygen species production without appreciably affecting their viability as determined by trypan blue staining. 0, from CS might cause this effect. To confirm this possibility, we measured O2 consumption of PMNs. The advantage of this measurement is that the activity of the respiratory burst can be monitored even in the presence of SOD, whereas PMN-induced chemiluminescence and DMPO spin adduct signals are almost quenched by SOD (40, 48). The protective effect of SOD on PMN 0, consumption indicates that CS-derived 0, is the initiating factor involved in impairment of PMNs, although the possibility that the oxidation products of CS components such as polyphenols which may be related with 0, generation (9, 12) may also damage PMN function has still not been eliminated. The similar protective effect by the iron chelator, DETAPAC, suggests that OH. generated from O- by iron catalysis (iron-catalyzed Fenton or Haber-Weiss reaction) (49) are involved in the toxicity of CS exposure. Despite the fact that PMNs are one of the major sources of active oxygen production such as 0; in uiuo, the finding that PMNs are impaired when 0, is exogenously generated in their suspended buffer is not very surprising. Presumably the various antioxidant systems in PMNs provide defense against the oxidative stress produced by their own respiratory burst (50). However, t,heir endogenous defenses may not be completely adequate and may be overwhelmed by exogenous oxidative stress (50). Our results also show that the PMN respiratory burst can be damaged by 0, generation in PMN suspensions by the reaction of xanthine with xanthine oxidase. Since PMNs can continuously produce 0, once activated (51), it may be that intracellular signal transduction processes which trigger the respiratory burst may be quite susceptible to oxidative stress. As the CS-derived 0, in the buffer proved to be a primary cause of PMN impairment, it was of interest to
36
TSUCHIYA
investigate the possible protective effect of antioxidants like a-tocopherol (52), P-carotene (53), and DHLA (54), which are known to be effective natural antioxidants in viuo and in vitro. Earlier reports have indicated that the presence of an excess of these antioxidants modifies the 0; production by PMNs (55-59). However, PMNs which were previously treated with small amounts of antioxidants became comparatively resistant to CS exposure without modification of their active oxygen generation ability. The marked effect of a-tocopherol is in agreement with the finding that it is the most effective lipid-soluble antioxidant in protecting cell membranes against oxidative damage (60). Although the mechanisms of G-related lung diseases are complex, it is conceivable that 0; from CS has a role in the development of these diseases, either by direct toxicity or by their effects on impairment of PMN activity. Hence prior dietary supplementation with antioxidants such as a-tocopherol could be effective in protecting against the deleterious effects of CS exposure. ACKNOWLEDGMENTS This research was supported by funds provided by the Cigarette and Tobacco Surtax Fund of the State of California through the TobaccoRelated Disease Research Program of the University of California, Grant IRT28, and Asta Medica. The authors thank Drs. Barry Halliwell (Davis), Valerian E. Kagan (Berkeley), William A. Pryor (Baton Rouge), and Kozo Utsumi (Kochi) for valuable suggestions.
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ET AL. Chemistry, Biology and Medicine Elsevier, Amsterdam. 15. Nakayama, T., Kaneko, Nature 3 14,462-464.
(Rotilio,
G., Ed.), pp. 467-472,
M., Komada, M., and Nagata, C. (1985)
16. Borish, E. T., Pryor, W. A., Venugopal, (1987) Curcinogenesis 8, 1517-1520.
S., and Deutsch,
W. A.
17. Frei, B., Forte, T. M., Ames, B. N., and Cross, C. E. (1991) Biochem. J. 277,133-138. 18. Babior, B. M. (1978) N. Engl. J. Med. 298, 659-668. 19. Halliwell, B., and Gutteridge, J. M. C. (1989) in Free Radicals in Biology and Medicine, 2nd ed., pp. 372-390, Clarendon, Oxford. 20. Bergstrand, H., Bjornson, A., Eklund, A., Hernbrand, R., Larsson, K., Linden, M., and Nilsson, A. (1986) J. Free Radicals Biol. Med. 2,119-127. 21. Beswick, P. H., Brannen, P. C., and Hurles, S. S. (1986) J. Clin. Lab. Immunol. 21, 71-75. 22. Gillespie, M. N., Owasoyo, J. O., Kojima, S., and Jay, M. (1987) Toxicology 45,45-52. 23. Abtsmd, W. R., Kucich, U., Kimbel, P., Glass, M., and Weinbaum, G. (1988) Exp. Lung Res. 14,459-475. 24. MacNee, W., Wiggs, B., Belzberg, A. S., and Hogg, J. C. (1989) N. Eng1.J. Med. 321,924-928. 25. Mccusker, 682.
K., Hoidal,
J. (1990) Am. Rev. Respir. Dis. 141, 678-
26. Kiyosawa, H., Suko, M., Okudara, H., Murata, K., Miyamoto, T., Chung, M., Kasai, H., and Nishimura, S. (1990) Free Radical Res. Commun. 11, 23-27. 27. Brown, G. M., Drost, E., Donaldson, K., MacGregor, I., and MacNee, W. (1991) Exp. Lung Res. 17,923-937. 28. Green, G. M. (1985) Eur. J. Respir. Dis. Suppl. 139,40-48. 29. Green, G. M. (1985) Eur. J. Respir. Dis. Suppl. 139, 4113-4116. 30. Voisin, C., Aerts, E., and Firlik, F. M. Eur. J. Respir. Dis. Suppl.
139,76-81. 31. Codd, E. E., Swim, A. T., and Bridges, R. B. (1987) J. Lab. Clin. Med. 110,648-652. 32. Butler, J., and Halliwell, B. (1982) Arch. Biochem. Biophys. 218, 174-178. 33. Rahhal, S., and Richter, W. (1988) J. Am. Chem. Sot. 110, 31263133. 34. Tsuchiya, M., Okimasu, E., Ueda, W., Hirakawa, M., and Utsumi, K. (1988) FEBS Lett. 242, 101-105. 35. Utsumi, K., Sugiyama, K., Miyahara, M., Naito, M., Awai, M., and Inoue, M. (1977) Cell Struct. Funct. 2, 203-209. 36. Fallon, H. J., Frei, III, E., Davidson, J. D., Trier, J. S., and Burk, D. (1962) J. Lab. Clin. Med. 59, 779-791. 37. Schlager, S. I., and Adams, A. C. (1983) in Methods in Enzymology (Langone, J. J., and Vunakis, H. V., Eds.), Vol. 93, pp. 233-245, Academic Press, New York. 38. Sato, E., Takehara, Y., Sasaki, J., Matsumoto, T., and Utsumi, K. (1986) Cell Struct. Funct. 11, 125-134. 39. Corbisier, P., Houbion, A., and Remacle, J. (1987) Anal. Biochem.
164,240-247. 40. Allen, R. C. (1986) in Methods in Enzymology (Deluca, M. A., and McElroy, W. D., Eds.), Vol. 133, pp. 449-493, Academic Press, New York. 41. Matsumoto, T., Orita, K., Sato, E., Nobori, K., Inoue, B., and Utsumi, K. (1987) Biochem. Phurmacol. 36, 1613-1616. 42. Black, C. D. V., Samuni, A., Cook, J. A., Krishna, C. M., Kaufman, D. C., Maech, H. L., and Russo, A. (1991) Arch. Biochem. Biophys. 286,126-131. 23, 239-257. 43. Fridovich, I. (1983) Annu. Reu. Phurmucol. Toxicol.
SUPEROXIDE
FROM
CIGARETTE
44. Fridovich, I. (1986) Arch. Biochem. Biophys. 247, l-11. 45. Sawyer, D. T., and Valentine, J. S. (1981) Act. Chem. Res. 14,393400. 46. Frimer, A. A. (1983) in The Chemistry
of Functional Groups, Peroxides (Patai, S., Ed.), pp. 429-461, Wiley, New York.
47. Halliwell,
B., and Gutteridge, J. M. C. (1989) in Free Radicals in Biology and Medicine, 2nd ed., pp. 22-85, Clarendon, Oxford.
48. Ueno, I., Kohno, M., Mitsuta, K., Mizuta, (1989) J. Biochem. 105,905-910.
Y., and Kaegasaki,
Stress (Sies, H.,
Ed.), pp. 351-382, Academic Press, New York.
37
Biophys. 282, 221-225. L. (1992) in Methods in Enzymology (Packer, L., Ed.), Vol. 213, pp. 460-472, Academic Press, New York. 54. Suzuki, Y. J., Tsuchiya, M., and Packer, L. (1991) Free Radical Res.
Commun. 15,255-263. 55. Baehner, R. L., Boxer, L. A., Ingraham, L. M., Butterick,
Cn a I.
58. 59.
51. Samuni, A., Krishna,
C. M., Cook, J., Black, C. D. V., and Russo, A. (1991) Free Rad. Biol. Med. 10, 305-313.
NEUTROPHILS
53. Tsuchiya, M., Scita, G., Freisleben, H. J., Kagan, V. E., and Packer,
56.
B., and Gutteridge, J. M. C. (1989) in Free Radicals in Biology and Medicine, 2nd ed., pp. 160-170, Clarendon, Oxford.
IMPAIRS
52. Kagan, V. E., Serbinova, E. A., and Packer, L. (1991) Arch. Biochem.
S.
49. Halliwell,
50. Hamers, M. C., and Roos, D. (1985) in Oxidative
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60.
C., Haak, R. A. (1982) Ann. N.Y. Acad. Sci. 293, 237-250. Weiamn, S. J., Lafuze, J. E., Haak, R. A., and Baehner, R. L. (1987) Inflammation 11, 309-321. Chan, A. C., Tran, K., Pyke, D. D., and Powell, W. S. (1989) Biochim. Biophys. Acta 1005, 265-269. Boxer, L. A. (1990) Adu. Exp. Med. Biol. 262, 19-33. Anderson, A., and Theron, A. J. (1990) in World Review of Nutrition and Dietetics (Bourne, G. H., Ed.), Vol. 62, pp. 27-58, Karger, Basel. Packer, L. (1991) Am. J. Clin. N&r. 53, 105OS-1055s.
OF BIOCHEMISTRY
Vol. 299, No. 1, November
AND
BIOPHYSICS
15, pp. 30-37,1992
Superoxide Formed from Cigarette Smoke Impairs Polymorphonuclear Leukocyte Active Oxygen Generation Activity Masahiko
Tsuchiya,’
David
F. T. Thompson,
Yuichiro
J. Suzuki,
Carroll
E. Cross,*
and Lester
Packer
Department of Molecular and Cell Biology, 251 LSA, University of California, Berkeley, California 94720; and *Department of Medicine and Physiology, University of California, Davis, California 95616
Received April 1, 1992, and in revised form July 21, 1992
Reactive free radicals contained in cigarette smoke (CS) and compromised phagocytic antimicrobial activities including those of polymorphonuclear leukocytes (PMNs) have been implicated in the pathogenesis of severe CS-related pulmonary disorders. In CS-exposed buffer solutions, 0, was the predominant generated reactive oxygen species, as demonstrated by lucigenin-amplified chemiluminescence and electron spin resonance (ESR) spin-trapping with 5,5-dimethyl-1-pyrroline Noxide (DMPO). When PMNs were incubated in this buffer, phorbol 12-myristate 13-acetate (PMA)-stimulated active oxygen production and coupled O2 consumption were strongly impaired without appreciably affecting PMN viability (1-min exposure inhibited active oxygen production by 75%). Superoxide dismutase (SOD) totally protected and an iron chelator, diethylenetriaminepentaacetic acid (DETAPAC), also protected the CS-exposed PMNs, suggesting that generated 0; was an initiating factor in the impairment and OH - generation was a subsequent injurious factor. Pretreatment of PMNs with antioxidants such as a-tocopherol and dihydrolipoic acid (DHLA) was partially protective. The results suggest that (i) 0; is probably generated in the upper and lower respiratory tract lining fluid when they come in contact with CS; (ii) such generated 0; can primarily impair PMN capabilities to generate reactive oxygen species; and (iii) since these effects may contribute to the pathogenesis of CS-related lung diseases, prior supplementation with antioxidants such as a-tocopherol or DHLA might be successful in preventing these deleterious effects. kc) 1992 Academic Press, Inc.
Cigarette smoking is associated with pulmonary disorders including predisposition for respiratory infections, 1 To whom correspondence
should be addressed.
pulmonary emphysema, and cancer (l-4). Although the exact mechanisms by which smoking produces these effects are incompletely understood, it has been hypothesized that the free radicals contained in or generated by CS2 may be among the potential causative factors (4, 5). The free radical species contained in CS have been extensively studied by Pryor, Church, and their coauthors (6-10). They reported various kinds of free radicals derived from CS itself, which are possibly toxic. To estimate the toxicity of these radicals in uiuo, however, radical species which are generated in CS-exposed aqueous solution should be also considered, because inspired CS first interacts with aqueous surfaces of the upper and lower respiratory tract lining fluid. OH * generation from aqueous extract of tar (11) and H202 generation in the CS-bubbled aqueous solution (12) have already been reported by Pryor and Nakayama, respectively. Pryor et al. provide evidence that 0; was generated as an intermediate in this reaction (11, 13-14). In addition, it has been demonstrated that these radicals induce single-strand breaks of DNA (1316). It was also reported by Cross et al. that the interaction of CS with plasma induced lipid peroxidation (17). Alveolar macrophages and PMNs are thought to play an essential role in antimicrobial defense of the lung. Once coming across noxious agents, these cells produce a flood of bactericidal reactive oxygen species, including O,, OH -, and HOCl/OCl-, a phenomenon known as the NADPH oxidase-mediated respiratory burst (18, 19). Several studies have suggested that smoking causes qualitative and quantitative alterations in these inflammatory ’ Abbreviations used: CS, cigarette smoke; DETAPAC, diethylenetriaminepentaacetic acid; DHLA, dihydrolipoic acid, DMPO, 5,5-Dimethyl-1-pyrroline N-oxide; ESR, electron spin resonance; hfc, hyperfine coupling constant; KRP, Krebs-Ringer-phosphate; lucigenin, lO,lO’-dimethyl his-9,9’-biacridinium dinitrate; luminol, 5-amino-2,3-dihydro-1,4phthalazinedione; PMA, phorbol-12-myristate-13-acetate; PMNs, polymorphonuclear leukocytes; SOD, superoxide dismutase.
30
000%9861/92 All
$5.00
Copyright 8 1992 by Academic Press, Inc. rights of reproduction in any form reserved.
SUPEROXIDE
FROM
CIGARETTE
phagocytic cells (20-26), which possibly increase the susceptibility to infection or damages airway alveolar and/ or lining fluid components involved in respiratory tract defense mechanisms. This may be an important process in the development of several CS-related diseases. Thus, the effects of CS on the functions of these phagocytes are of importance. Reduction of proteolytic activity (27), inhibition of chemotaxis (28), decrease in phagocytic and bactericidal activity (29, 30), and desensitization to chemotactic peptide (31) of macrophages and PMNs have been reported as in vitro primary effects of CS. However, to our knowledge, few studies have been focused on the direct effect of CS on the generation of antimicrobial (and potentially injurious) reactive oxygen species from these inflammatory and phagocytic cells. Therefore, the goal of the present research was to study the radical generation in CS-exposed aqueous solutions, and to determine whether CS exposure affects the respiratory burst of PMNs and whether CS-derived radicals play a role in the process. MATERIALS
AND
METHODS
Chemicals DMPO was purchased from Daiichi Pure Chemicals Co., Ltd. (Tokyo). Lucigenin, luminol, PMA, SOD (from bovine erythrocytes), and catalase (from bovine liver) were from Sigma Chemical Co. (St. Louis, MO). Xanthine oxidase from cow’s milk was from Boehringer Mannheim Biochemicals (Indianapolis, IN). The University of Kentucky (UK) 2RI standard cigarettes containing 23 mg tar and 2.2 mg nicotine per cigarette (according to the U.S. Federal Tobacco Council) were used. a-Tocopherol was a gift from Henkel Corp. (LaGrange, IL), DHLA was from Asta Medica (Frankfurt), and p-carotene was from Roche Vitamins and Fine Chemicals. (Nutley, NJ). Other reagents were commercial products of analytical grade.
Measurement Solution
of Radical Generation from CS in Aqueous
Chem&minescence assay. Unfiltered main stream CS from the standard cigarette was bubbled through a 20 mM phosphate buffer (pH (32, 33) for 7.4) containing 100 mM lucigenin and 0.1 mM DETAPAC 2 min at a flow rate of 50 ml/min using the method of Pryor et al. (7) (Fig. 1). After the termination of bubbling, lucigenin-sensitive chemiluminescence of the buffer was recorded with a luminometer (LKB Wallac 1250, Finland). ESR spin trapping technique. Radical generation in the aqueous soluticn was also measured by the ESR spin trapping technique using the cyclic nitrone spin trap, DMPO. A DMPO solution (500 mM) with 0.5 mM DETAPAC was bubbled with the main stream CS for 5 min at a flow rate of 50 ml/min, as shown in Fig. 1. The solution was transferred to a flat quartz ESR cuvette (0.3.mm-thick flat cell, LABOTEC, Japan), which was placed in the cavity of the ESR spectrometer (IBM ER2OOD. SRC). Recordings of the spectra were made at modulation amplitude 1.25 G, modulation frequency 100 kHz, microwave power 9.6 mW, time constant 0.5 s, receiver gain 1 X 106, and sweep time 30 G/min. The hfc were measured directly from the scan field using MnO as an external standard. 0, spin adduct. The usual spectra of a 0; spin adduct of DMPO were recorded by the reaction of 100 mu/ml xanthine oxidase with 5 mM xanthine in a 30 mM phosphate buffer (pH 7.4), containing 100 mM DMPO and 0.1 mM DETAPAC.
SMOKE
IMPAIRS
FIG. 1.
31
NEUTROPHILS
System for exposure of CS to samples.
Measurement of PMNs Respiratory Burst and Effect of CS Exposure Preparation of PMNs. PMNs were isolated at 4°C from the peritoneal cavity of male Sprague-Dawley rats (250-300 g) 12 h after the intraperitoneal injection of 2% neutrose (2% neutralized casein sodium in norma! saline (pH 7.4)) as previously described (32,33). The isolated PMNs were suspended in KRP buffer, consisting of 154 mM NaCl, 6 mM KCl, 1 mM MgCl,, and 10 mM sodium phosphate buffer (pH 7.4). All measurements of PMNs were finished within 3 h after the start of the isolation. Measurementof active oxygen production. Production of reactive oxygen species from PMNs was measured by chemiluminescence and ESR spin trapping techniques. PMNs (2 X lo6 cells/ml) were incubated in a KRP buffer with 5 mM glucose, 500 pM calcium, and 100 pM lucigenin (or luminol) (for the chemiluminescence assay) or 50 mM DMPO (for the ESR spin trapping assay) and stimulated by the addition of 10 nM PMA. The chemiluminescence assays were performed using a luminometer, whereas the spin adduct of DMPO was measured with an ESR spectrometer using the same settings described above. Measurement of 0, consumption. 0, consumption coupled with the respiratory burst was measured polarographically with a Clark-type oxygen electrode. PMNs (1 X lo7 cells/ml) were incubated in KRP buffer with 10 mM glucose and 1 mM calcium and stimulated by 100 nM PMA. Measurement of cell uiability. PMN suspension was mixed with trypan blue (0.02% in final concentration) (36, 37). Since unviable cells could not exclude dye, the percentage of cells stained with dye is a measure of viability. Effect of CS on PMNs. PMNs in a KRP buffer were bubbled with main stream CS at a flow rate of 50 ml/min for 1 min as shown in Fig. 1; then active oxygen generation (chemiluminescence and ESR measurement), 0, consumption, and viability of PMNs were measured. In the case of chemiluminescence and ESR measurements, lucigenin (or luminol) or DMPO was added to the PMN suspension after the termination of exposure, which served to minimize or eliminate CS-derived free radical signals from PMN-derived active oxygen signals. Effect of 0; generated by ranthine and xanthine oridase on PMNs. After 2-min exposure to 0; generated by the reaction of 10 mu/ml xanthine oxidase with 50 FM xanthine, PMNs (2 X lo6 cell/ml) were stimulated by PMA, and lucigenin-amplified chemiluminescence was measured.
Protective Effect of Antioxidants
on CS Exposure
PMNs were preincubated for 5 min with various antioxidants (LYtocopherol, p-carotene, and DHLA) individually in order to allow incorporation of the antioxidant into the PMNs, and were washed with
32
TSUCHIYA
KRP buffer two times (38). These pretreated PMNs in a KRP buffer were bubbled by main stream CS at flow rate of 50 ml/mm for 1 min as shown in Fig. 1, and then their active oxygen generation activity induced by PMA was measured by lucigenin chemiluminescence and ESR spin trapping.
ET AL.
(A) MODEL SYSTEM XANTHINE +XANTHINE OXIDASE
Temperature and Oxygen Control All reactions were carried out at 37°C. Chemiluminescence and 0, consumption were measured using a thermostated cuvette equipped with a mixing device which maintained relatively homogeneous PMN suspension and supplied 0, to the PMNs from the atmosphere during the measurement. ESR spectra were recorded in a cell maintained thermostatically by nitrogen gas flow.
(8) SMOKE AQUEOUS
EXPOSED SOLUTION
Data Processing All experiments were repeated three to five times, and data are expressed as means f SD. The statistical significance of difference between two mean values was tested by Student’s t test.
RESULTS
Radical Generation from CS in Aqueous Solution CS exposure of phosphate buffer produced lucigenindependent chemiluminescence which is known to be highly sensitive to the 0; (39) (Fig. 2). The intensity of chemiluminescence gradually decreased after the termination of exposure. This chemiluminescence was totally eliminated by the addition of 100 units/ml SOD, indicating that 0; generation occurred in the CS-exposed buffer. The presence of 0; was confirmed by ESR spin trapping with DMPO. A typical DMPO-OOH (0, spin adduct of DMPO) adduct spectrum was generated by the xan-
I (C) pretreated with (D) AIR EXPOSED I
I
I
I SOD 01
I
T
(Cl EXPOSURE + SOD
FIG. 3. ESR spectra of spin adducts generated by CS exposure of aqueous solution in the presence of DMPO. Reaction mixture (300 ~1, 37°C) contained 0.5 mM DETAPAC in 500 mM DMPO solution before CS exposure. (A) Standard spectra of the 0; spin adduct of DMPO generated by 100 munits/ml xanthine oxidase plus 5 mM xanthine in 30 mM phosphate buffer (pH 7.4) containing 50 mM DMPO and 0.1 mM DETAPAC. (B) DMPO spin adduct spectra of 5-min CS-bubbled solution in the absence of SOD. (C) DMPO spin adduct spectra of 5-min CS-bubbled solution in the presence of 100 units/ml SOD at the beginning of CS exposure.
thine and xanthine oxidase system, and an almost identical spectrum (aN, 14.3 G; a;, 11.5 G; a:, 1.3 G) was observed in the CS-exposed DMPO solution in the company of a small amount of DMPO-OH (OH. spin adduct of DMPO: a,“, 14.8 G; a,!j, 14.8 G) (Fig. 3). The signal was not detected in the presence of 100 units/ml SOD or when the DMPO solution was bubbled with air in place of CS. Effect of CS Exposure on Reactive Oxygen Species Production by PMNs
I
6 4 2 INATION CFEQTSBE TIME IN MINUTES
SMOKE
8
FIG. 2. Lucigenin-dependent chemiluminescence generated in CSbubbled buffer. Reaction mixture (2 ml, 37’C) contained 100 mM lucigenin and 0.1 mM DETAPAC in 20 mM phosphate buffer (pH 7.4) before CS exposure. (A) 2-min CS-bubbled buffer. (B) 2-min CS-bubbled buffer + 100 units/ml SOD as indicated. (C) 2.min CS-bubbled buffer in the presence of 100 units/ml SOD at the beginning of CS exposure. (D) Control (2.min air-bubbled buffer).
After treatment with PMA, PMNs produce various reactive oxygen species, which can be monitored by either lucigenin or luminol-sensitive chemiluminescence (40). The chemiluminescence of both lucigenin and luminol was equally inhibited by exposure to CS in an exposureduration-dependent manner (Figs. 4 and 5), being almost totally inhibited by a 5-min CS exposure. The O2 concentration in PMN suspension in control and CS exposure was maintained at about 220 PM with a variation of 20 PM during measurement. Effect of CS Exposure on O2 Consumption
of PMNs
The respiratory burst of the PMNs is accompanied by a remarkable O2 consumption (41) (Fig. 6), which is an another reliable index of the PMN active oxygen gener-
SUPEROXIDE
6
n
WITH LUCIGENIN
A
WITH LUMINOL
E
8
FROM
CIGARETTE
60
B
SMOKE
IMPAIRS
erated, did not respond fully to the PMA stimulation (Fig. 7). The magnitude of the chemiluminescence response was less than half of that of PMNs which were not exposed to 0;. In control the presence of either 20 PM xanthine or 10 PM uric acid, the final product of xanthine oxidation, did not affect chemiluminescence generation by PMNs, and the inhibitory effect by 0, generation in the suspension was not affected in the presence of 8800 units/ml catalase, which eliminated another final product, Hz02, of xanthine oxidation. The O2 concentration in the PMN suspension was maintained at about 220 PM with a variation of 20 PM during measurement. Effect of Antioxidants
SMOKE EXPOSURE
TIME (SEC)
FIG. 4. Time dependent effect of CS exposure on PMNs respiratory burst measured by chemiluminescence. The reaction mixture (2 ml) contained 2 X 10s cells/ml PMNs, 154 mM NaCl, 6 mM KCl, 1 mM MgCl,, 5 mM glucose, and 500 FM CaCl, in 10 mM sodium phosphate buffer (pH 7.4) at 37°C. PMNs were stimulated by 10 nM PMA and their responses were estimated by measuring the area of generated chemiluminescence signal for 8 min. Closed square, chemiluminescence amplified by 100 pM lucigenin. Open triangle, chemiluminescence amplified by 100 FM luminol.
ation activity. Exposure of PMN suspension to CS reduced O2 concentration in the suspension by less than 10% of control and this reduction immediately recovered during the process for transfer of sample from CS exposure apparatus to oxygen electrode. The rate of O2 consumption in PMN activation was reduced by CS exposure, which agrees well with chemiluminescence results that CS impaired the respiratory burst. The presence of 100 units/ml SOD in the buffer markedly protected PMNs against CS exposure. SOD alone decreased the O2 consumption rate only slightly, possibly indicating enhanced dismutase reaction by exogenous SOD. The iron chelator, 10 PM DETAPAC (32, 33), which had no effect of PMN O2 consumption, also protected PMNs from the CS-induced inhibition.
33
NEUTROPHILS
on the CS Exposure
Based on the above results, it is expected that antioxidants might protect PMNs against smoke exposure. To estimate this protective effect, we determined how well the respiratory burst was maintained after CS exposure. The degree of effectiveness varied according to the antioxidant incorporated (Table II). In particular, cr-tocopherol-treated PMNs showed a good resistance to CS exposure (Fig. 5). Pretreatment of PMNs with the concentrations of antioxidants used did not affect active oxygen production of PMNs (data not shown). The protective effect of ol-tocopherol was also confirmed by spin trapping. The DMPO-OOH (DMPO-superoxide) signal followed by small DMPO-OH (DMPO-hydroxy radical) was observed in PMA-stimulated intact PMN suspension (42) (Fig. 8). CS exposure reduced these signals in untreated PMNs, but was only slightly affected in (Ytocopherol-pretreated PMNs. For measurement of the
Effect of CS Exposure on Cell Viability The percentage of PMNs stained with trypan blue did not increase significantly after 5 min exposure to CS at the highest dose used in any of our experiments (Table I). 6
Effect of Enzyme-Generated
0; on PMNs
To confirm toxicity of 0; generated in PMN suspension, 05 was enzymatically generated in the suspension under the same conditions as those of CS exposure. PMNs, which were previously incubated for 2 min in the buffer, where small amounts of 0; comparable to those generated in CS-exposed buffer were enzymatically gen-
TIME IN MINUTES FIG. 5. Effect of CS exposure on active oxygen generation by PMAstimulated PMNs. Active oxygen generation was monitored with lucigenin-sensitive chemiluminescence. PMNs were stimulated by 10 nM PMA. Conditions as described in the legend to Fig. 4 in the presence of 100 pM lucigenin. (A) Control (air exposure). (B) 1-min CS exposure to PMNs pretreated by 2.5 ~.LM a-tocopherol. (C) l-min CS exposure to PMNs.
34
TSUCHIYA
ET AL.
XANTHINE
0
(D) SMOKE EXPOSURE
/
8
4
12
16
0
TIME IN MINUTES I 0
f
1
I
4
a
12
I 16
20
TIME IN MINUTES FIG. 6. Effect of CS exposure on 0, consumption by PMNs coupled with active oxygen generation. Conditions as described in the legend to Fig. 4 except for concentration of PMN (1 X lo7 cells/ml) CaCl, (1 mM), glucose (10 mM) and PMA (100 nM). (A) Control (air exposure). (B) Air exposure in the presence of 10 PM DETAPAC at the beginning of exposure. (C) Air exposure in the presence of 100 units/ml SOD at the beginning of exposure. (D) 1.5.min CS exposure in the presence of 100 units/ml SOD at the beginning of exposure. (E) 1.5.min CS exposure in the presence of 10 HIM DETAPAC at the beginning of exposure. (F) 1.5-min CS exposure.
respiratory burst with ESR, DMPO was added to a PMN suspension after the termination of CS exposure, since this method of procedure allows the elimination or minimization of any contamination by CS-produced ESR signals. As a result, no ESR signal was observed in unstimulated CS-exposed PMN suspension, and therefore the signal recorded after PMN stimulation is considered to specifically represent reactive oxygen species generated by PMNs.
FIG. 7. by PMNs. of 100 pM xanthine nM PMA. enzymatic
Effect of enzyme-produced 0; on active oxygen generation Conditions as described in the legend to Fig. 3 in the presence lucigenin. 0; was generated by the reaction of 10 mu/ml oxidase with 50 pM xanthine. PMNs were stimulated by 10 (A) Control (no exposure to 0;). (B) Exposure to 0; generated reaction.
DISCUSSION
CS-related respiratory tract diseases are generally recognized to represent the combined effects of direct CS injury to the cellular component of respiratory tract and secondary inflammatory immune and infectious process related to the primary CS damage. Presumably, both CS and phagocytic reactive oxygen species play a role in this process. The role of CS oxidant is complex because of the multitude of free radical species known to be present in CS (7, 9), whereas the role of phagocytic oxidant is theo-
TABLE Effect
of Antioxidants of PMNs
Treatment TABLE Effect
of CS on Tryptan
II
on the Respiratory Exposed to CS
Burst
Chemiluminescence intensity (arbitrary unit)
I
Blue Dye Exclusion
in PMNs
Treatment
Cells strained with trypan hlue (percentage)
Control (air exposure) CS exposure (5 min)
2.0 f 1.4 5.9 k 2.6
Note. PMN suspension (containing 2 X 10” cells/ml PMNs, 154 mM NaCI, 6 mM KCl, 1 mM MgCl,, 5 mM glucose, and 500 FM CaCl* in 10 mM sodium phosphate buffer (pH 7.4, at 37°C) was bubbled by main stream of CS at a flow rate of 50 ml/min for 5 min as in Fig. 1, and diluted with trypan blue (0.02% in final concentration), and then the number of PMNs stained with dye was counted. The percentage was recorded by at least eight separate countings.
Control (air exposure) CS exposure + n-Tocopherol t DHLA /j-Carotene
100.0 20.0 t 0.4 47.0 i 5.4* 47.3 + 4.3-3
31.8 * 2.1*
Note. PMNs were preincubated for 5 min with 2.5 /rM of the antioxidant indicated, and exposed to main stream of CS at a flow rate of 50 ml/min for 1 min as in Fig. 1, and then stimulated with 10 nM PMA at 37°C. The respiratory burst of PMNs was monitored by measuring lucigenin-amplified chemiluminescence. Control measurements were performed with PMNs preincubated with solvent alone. The reaction mixture contained 2 X lo6 cells/ml PMNs, 154 mM NaCl, 6 mM KCl, 1 mM MgCl,, 5 mM glucose, 500 pM CaClz, and 100 fiM lucigenin in 10 mM sodium phosphate buff&r (pH 7.4). Each response of PMNs was estimated by measuring the area of chemiluminescence signal for 8 min. * Significantly different from CS exposure at P < 0.05.
SUPEROXIDE
FROM
CIGARETTE
(A) CONTROL
(B) SMOKE
/
EXPOSURE
ICI SMOKE
EXPOSURE
a-TOCOPHEROC TREATED PMNs
FIG. 8. Protective effect of n-tocopherol on CS-exposed PMNs. PMNs were stimulated by 10 nM PMA, and generated active oxygen was monitored by DMPO (50 mM) spin adduct formation 8 min after the stimulation. Conditions as described in the legend to Fig. 4. (A) Control (air exposure). (B) l-min CS exposure to PMNs. (C) I-min CS exposure to PMNs pretreated by 2.5 pM cu-tocopherol.
retically both adventitious (antimicrobial activity) and injurious (capable of oxidatively injuring neighboring cells including phagocytes). The present studies detailed some of the interactions of these two potent oxygen radical generating systems believed to be relevant when both are present in respiratory tract lining fluids; we focused especially on the known generation of 0, by both CS and PMNs and the possible consequences of their interaction. 0; is not only toxic itself (43, 44), but it also would lead to the generation of additional potentially and highly toxic metabolites such as H,O,, OH *, and ‘0, (45-47). If such 0, is formed at the surfaces of the upper and lower respiratory tract lining fluid when it comes in contact with CS, surrounding airway and lung parenchymal (alveolar) components, which form a mechanical barrier against microorganisms, may be seriously damaged. Thus damage may increase both airways and lung parenchymal susceptibility to infection. PMNs are an essential component of antimicrobial defenses of the lung. There have been several reports that there is a significant increase in the number of PMNs and/or the oxygen-derived free radicals from PMNs in the plasma and alveolar lavage fluid of cigarette smokers or CS-exposed animals (20, 22-24). The observed effect is interpreted as an activation effect of CS on PMNs, which conceivably leads to oxidative damage to airway or to alveoli. However, the “influx” and “activation” of PMNs into the lung may he due to the effect of release of chemical mediators secondary to tissue damage induced by CS, and the localized and primary effect of CS on phagocytes might then be masked. According to the tox-
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IMPAIRS
NEUTROPHILS
35
icity of CS (30), PMNs present in the respiratory tract lining fluids which are directly exposed to CS might themselves be injured. It has been reported that CS directly inhibited PMN chemotaxis (28, 31), phagocytosis (29), and proteolytic (27) and bactericidal (30) activity in vitro. The regional failure of these inflammatory cells in CS-threatened airways may be more important in the development of CS-related lung disease. It has been shown by Pryor et al. that tar-derived radicals in CS had a capacity for producing 0, in aqueous solution and that ESR spin adduct signals from cigarette tar is attenuated by the SOD (9, 11, 13, 14). Nakayama et al. have also shown that Hz02 is formed when CS is bubbled through water, suggesting that the 0; is an intermediate (12). In t,he present study with chemiluminescence and ESR, it is directly demonstrated that 0; was generated in the CS-exposed aqueous solution, which confirmed Pryor’s previous findings (9, 11, 13, 14). A brief exposure of PMNs to CS inhibited their reactive oxygen species production without appreciably affecting their viability as determined by trypan blue staining. 0, from CS might cause this effect. To confirm this possibility, we measured O2 consumption of PMNs. The advantage of this measurement is that the activity of the respiratory burst can be monitored even in the presence of SOD, whereas PMN-induced chemiluminescence and DMPO spin adduct signals are almost quenched by SOD (40, 48). The protective effect of SOD on PMN 0, consumption indicates that CS-derived 0, is the initiating factor involved in impairment of PMNs, although the possibility that the oxidation products of CS components such as polyphenols which may be related with 0, generation (9, 12) may also damage PMN function has still not been eliminated. The similar protective effect by the iron chelator, DETAPAC, suggests that OH. generated from O- by iron catalysis (iron-catalyzed Fenton or Haber-Weiss reaction) (49) are involved in the toxicity of CS exposure. Despite the fact that PMNs are one of the major sources of active oxygen production such as 0; in uiuo, the finding that PMNs are impaired when 0, is exogenously generated in their suspended buffer is not very surprising. Presumably the various antioxidant systems in PMNs provide defense against the oxidative stress produced by their own respiratory burst (50). However, t,heir endogenous defenses may not be completely adequate and may be overwhelmed by exogenous oxidative stress (50). Our results also show that the PMN respiratory burst can be damaged by 0, generation in PMN suspensions by the reaction of xanthine with xanthine oxidase. Since PMNs can continuously produce 0, once activated (51), it may be that intracellular signal transduction processes which trigger the respiratory burst may be quite susceptible to oxidative stress. As the CS-derived 0, in the buffer proved to be a primary cause of PMN impairment, it was of interest to
36
TSUCHIYA
investigate the possible protective effect of antioxidants like a-tocopherol (52), P-carotene (53), and DHLA (54), which are known to be effective natural antioxidants in viuo and in vitro. Earlier reports have indicated that the presence of an excess of these antioxidants modifies the 0; production by PMNs (55-59). However, PMNs which were previously treated with small amounts of antioxidants became comparatively resistant to CS exposure without modification of their active oxygen generation ability. The marked effect of a-tocopherol is in agreement with the finding that it is the most effective lipid-soluble antioxidant in protecting cell membranes against oxidative damage (60). Although the mechanisms of G-related lung diseases are complex, it is conceivable that 0; from CS has a role in the development of these diseases, either by direct toxicity or by their effects on impairment of PMN activity. Hence prior dietary supplementation with antioxidants such as a-tocopherol could be effective in protecting against the deleterious effects of CS exposure. ACKNOWLEDGMENTS This research was supported by funds provided by the Cigarette and Tobacco Surtax Fund of the State of California through the TobaccoRelated Disease Research Program of the University of California, Grant IRT28, and Asta Medica. The authors thank Drs. Barry Halliwell (Davis), Valerian E. Kagan (Berkeley), William A. Pryor (Baton Rouge), and Kozo Utsumi (Kochi) for valuable suggestions.
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