Free Radical Biology & Medicine, Vol. 12, pp. 365-372, 1992 Printed in the USA. All fights reserved.
0891-5849/92 $5.00 + .00 Copyright © 1992 Pergamon Press Ltd.
Original Contribution THE I N H I B I T O R Y
EFFECT OF EXTRACTS OF CIGARETTE TAR ELECTRON TRANSPORT OF MITOCHONDRIA AND SUBMITOCHONDRIAL PARTICLES
ON
WILLIAM A. PRYOR, NANCY C. ARBOUR, BRAD UPHAM, and DANIEL F. C H U R C H Biodynamics Institute, Louisiana State University, Baton Rouge, LA 70803, U.S.A. (Received 17 September 1991; Revised 30 December 1991 ; Accepted 2 January 1992)
Abstract--Acetonitrile extracts of cigarette tar inhibit state 3 and state 4 respiration of intact mitochondria. Exposure of respiring submitochondrial particles to acetonitrile extracts of cigarette tar results in a dose-dependent inhibition of oxygen consumption and reduced nicotinamide adenine dinucleotide (NADH) oxidation. This inhibition was not due to a solvent effect since acetonitrile alone did not alter oxygen consumption or NADH oxidation. Intact mitochondria are less sensitive to extracts of tar than submitochondrial particles. The NADH-ubiquinone (Q) reductase complex is more sensitive to inhibition by tar extract than the succinate-Q reductase and cytochrome complexes. Nicotine or catechol did not inhibit respiration of intact mitochondria. Treatment of submitochondrial particles with cigarette tar results in the formation of hydroxyl radicals, detected by electron spin resonance (ESR) spin trapping. The ESR signal attributable to the hydroxyl radical spin adduct requires the presence of NADH and is completely abolished by catalase and to a lesser extent superoxide dismutase (SOD). Catalase and SOD did not protect the mitochondrial respiratory chain from inhibition by tar extract, indicating that the radicals detected by ESR spin trapping are not responsible for the inhibition of the electron transport. We propose that tar causes at least two effects: ( l ) Tar components interact with the electron transport chain and inhibit electron flow, and (2) tar components interact with the electron transport chain, ultimately to form hydroxyl radicals. Keywords--Mitochondria, Cigarette tar, Electron transport, Autooxidation, Electron spin resonance, Spin trapping, Free radicals
mitochondria. Consistent with this hypothesis, exposure to cigarette tar has been shown to alter mitochondrial ultrastructure in cultured protozoa) ° Several studies have indicated that cigarette smoke inhibits mammalian mitochondrial function. 11:2 In one of the earliest reports, Kolberg x3 suggested that the depletion of oxygen by lung tissue homogenates was caused by cigarette-smoke-induced damage to mitochondrial membranes. Kyle et al. 14 demonstrated decreased phosphorylative efficiency in guinea pig lung mitochondria exposed to whole tobacco smoke in vivo. Several in vitro studies by Gairola et al. 15-17 showed that whole smoke alters respiration from pyridine-linked or ravin-linked substrates in intact mitochondria; depending on the protocol for the preparation of the tobacco smoke extract, either partial stimulation or inhibition of oxygen utilization by the mitochondria was observed. Attempts were made to localize the electron transport chain component(s) with which cigarette smoke interacted; spectroscopic measurement of the cytochromes in their reduced
INTRODUCTION
Over 4000 compounds, many of them highly toxic, have been identified in tobacco smoke.~-~ There is epidemiological evidence linking cigarette smoke with cancers of the larynx and lung, 4'5 and smoking has been suggested to be responsible for some or most of the deaths due to emphysema and chronic obstructive lung disease. 6,7Cigarette tar has been shown to possess redox properties that alter the activity of biologically important macromolecules. For example, Bilimoria et al. 8 have reported that tar accelerates the oxidation of ascorbate by a free-radical-generating reaction. Another study by these workers has shown that cigarette tar reduces cytochrome c,9 suggesting that tar might interact with and affect the electron transport chain of
Address correspondence to William A. Pryor and Daniel F. Church, Biodynamics Institute, Louisiana State University, Baton Rouge, LA 70803. 365
366
W.A. PRYOR et al.
state indicated that multiple cytochrome constituents are involved in the interaction of whole cigarette smoke and intact mitochondria. ~6 In the present study, we report an investigation of the mechanisms involved in cigarette-tar-mediated inhibition of mitochondrial electron transport and compare the effects in mitochondria isolated from beef heart and liver. We have incubated acetonitrile extracts of cigarette tar with intact mitochondria and submitochondrial particles (SMP) and followed both oxygen consumption and reduced nicotinamide adenine dinucleotide (NADH) oxidation. We also performed electron spin resonance (ESR) spin-trapping studies to ascertain whether oxy radicals are involved in the interaction of cigarette tar with SMP. MATERIALS AND M E T H O D S
Materials Research cigarettes (1RI series) were obtained from the Tobacco & Health Research Institute, University of Kentucky (Lexington, Kentucky). The nitrone spin trap, 5,5-dimethyl-l-pyrroline-N-oxide (DMPO), was purchased from Sigma Chemical Co. (St. Louis, MO) and purified with activated charcoal as described by Buettner and Oberley. 18 All other chemicals were used as supplied. Deionized water was used to prepare reagents. Buffers were treated with Chelex-100 to remove any contaminating iron. All reagents were prepared in water, except rotenone and antimycin, which were dissolved in ethanol and methanol, respectively.
Preparation of beef heart submitochondrial particles Fresh bovine hearts were obtained from a local slaughterhouse and were kept at 4°C throughout the procedure. Beef heart was minced into 1 in 3 pieces, and 200 g of this tissue were homogenized using a Waring blender in 0.25 M sucrose, 0.01 M Tris, pH 7.8. The pH of the resulting homogenate was adjusted to 7.8 with 6 M KOH. Mitochondria were isolated from the homogenate as described by Smith. 19 The mitochondria were resuspended in 250 mM sucrose, 1 mM succinate, 2 mM ethylenediaminetetraacetic acid (EDTA), l0 mM Tris, pH 7.8. After a 1-week storage at -20°C, the mitochondria were thawed and recentrifuged. The pellets were then homogenized in 100 mM sodium phosphate, pH 7.8. Submitochondrial particles were prepared in phosphate buffer according to Davies et al. 2° The Bradford method of protein determination was performed on the fresh preparation using bovine serum albumin as a stan-
dard. 21 Aliquots of the submitochondrial suspension were stored up to three months at -80°C.
Preparation of rat liver mitochondria Sprague-Dawley rats were fasted overnight and sacrificed by decapitation. The liver was removed and macerated with scissors in a buffer containing 210 mM mannitol, 70 mM sucrose, 5 mM HEPES/pH 7.4, 1 mM [ethylenebis-(oxyethylenenitrilo)]tetraacetic acid (EGTA), 1 mM EDTA, and 0.15% lipidfree bovine serum albumin. The macerated tissue was homogenized using a Potter-Elvehjem homogenizer with a teflon pestle. The homogenate was centrifuged at 200 × g for 10 min at 4°C. The supernatant solution was centrifuged at 6000 × g for 10 min at 4°C. The pellet was resuspended in a buffer containing 210 mM mannitol, 70 mM sucrose, and 5 mM HEPES/ pH 7.4. All assays with mitochondria were conducted in the resuspension buffer plus 5 mM MgCI2 and 5 mM potassium phosphate. Liver submitochondrial particles were prepared the same way as beef heart submitochondrial particles.
Cigarette tar preparation Standard conditions of storing and smoking the cigarettes were employed,z2,23 Prior to use, the cigarettes were conditioned at room temperature by storage over a saturated solution of ammonium nitrate. The tar from three cigarettes was collected on a Cambridge filter by drawing a 35-mL puffof smoke, 2 s in duration, once a minute through the filter. The filter was then placed in 7.5 mL of a solvent and allowed to soak for 5 min, the extract filtered and kept on ice. The solvents tested include acetonitrile, dimethyl sulfoxide (DMSO), and ethanol. To quantify the amount of cigarette tar collected, the filter was weighed after smoking the cigarettes (wet weight) and then after drying the filter completely (dry weight). On average, a weight corresponding to 22 mg of wet tar per cigarette (66 mg total from three cigarettes) was collected on the Cambridge filter. Quantitative measurements indicated that acetonitrile extracts approximately 40 mg of the 66 mg total wet tar (i.e., it extracts 13 mg/ cigarette). The other solvents tested appear to have an efficiency for extracting tar components that is very similar (see Table 1). The weight of tar described in the table and figure legends is the dry weight. While it cannot be assumed that all three solvents extract the same components from tar with the same efficiency, the tar extracts of all three appear to affect submitochondrial particles respiration similarly (see Table 1). Acetonitrile was used for most of our experiments be-
Effect of cigarette tar on electron transport Table 1. Extraction o f Cigarette Tar Components by Various Solvents: Effect on Oxygen Consumption by Beef Heart Submitochondrial Particles a Reaction Mixture NADH NADH NADH NADH NADH
+ + + +
Acetonitrile Tar-Ethanol Extract Tar-DMSO Extract Tar-Acetonitrile Extract
Rate b 151.0 137.0 23.0 21.0 18.0
+ 7.0 _+ 6.0 _+ 2.0 _ 4.0 _+ 2.0
a All treatments contained 100 m M sodium phosphate/pH 7.4. Concentrations of N A D H and protein arc 1.3 m M and 0.3 mg protein/mL, respectively. 50 uL of acetonitrile or tar extract containing approximately 300, 270, and 280 ug o f dry tar in ethanol, DMSO, and acetonitrile, respectively, was added per reaction mixture. Values are the average of two determinations. b Rate = nanomoles o f O 2 consumed/per minute per milligram of protein.
cause of its volatility and the ease of evaporating it from the Cambridge filters.
Respiration measurement in submitochondrial particles Respiration by submitochondrial particles was measured polarographically at 25°C with a Clarketype electrode (Yellow Springs Inst. Company, Yellow Springs, CO). Mitochondria were incubated in air-saturated medium containing 100 mM sodium phosphate, pH 7.8, giving a final volume of 3 mL. Either 1.3 mM NADH or 13 mM sodium succinate was used to initiate oxygen consumption.
NADH oxidation Oxidation of NADH was measured by following the absorbance decrease at 340 nm, using an extinction coefficient of 6.23 mM -~ cm-'. Absorbance changes were measured with a Hewlett Packard 8415A Diode Array Spectrophotometer. The reaction mixture contained submitochondrial particles (0.7 mg of protein/mL) in 1.0 mL of the medium used in respiration studies. After a 5-min incubation, tar extract was added followed with the addition of NADH.
ESR spectroscopy Spectra were recorded on a Bruker 100D X-band spectrometer equipped with an Aspect 2000 data acquisition system. The reaction mixture containing submitochondrial particles (3 mg/mL protein), 10 mM NADH, and 50 mM DMPO in 100 mM sodium phosphate, pH 7.8, was allowed to incubate for 1 min at room temperature. Tar extract was then added and
367
the sample mixed and purged with nitrogen. When included, scavengers were added prior to the addition of tar. The sample was transferred to a 17-mm quartz flat cell and degassed by vacuum before placing in the ESR cavity. Typically, three to five 200-s scans of each spectrum were recorded with a modulation frequency of 100 kHz, modulation amplitude 1.25 G, microwave power l0 mW, microwave frequency 7.4 kHz, and a scan rate of 30 G/min. RESULTS
Effects of extraction solvents Cigarette tar was collected on a Cambridge filter as described in "Materials and Methods," and several solvents were tested for their efficiency at extracting tar components from the filter. Table 1 presents a comparison of the rates of NADH-stimulated oxygen consumption by SMP from beef heart treated with ethanol, DMSO, and acetonitrile extracts of cigarette tar. The data (expressed as nanomoles of oxygen consumed per minute per milligram of protein) indicate that similar inhibited rates of respiration are observed for submitochondrial particles in the presence of tar extracts in any of these three solvents. The addition of acetonitrile to respiring SMP did not alter oxygen consumption; thus, the observed inhibition by tar extracts is not the result of a nonspecific effect of the solvent. Therefore, we adopted acetonitrile as a solvent for subsequent experiments because of its ease of evaporative removal. The effect of acetonitrile extracts of cigarette tar on NADH-stimulated respiration in rat liver SMP also inhibited respiration to the same extent (Tables 1 and 2), indicating that the source of the SMP made no difference. Exposure of rat liver submitochondrial particles to acetonitrile extracts of cigarette tar results in dose-dependent inhibition of both oxygen consumption and NADH oxidation, with maximal inhibition occurring at approximately 250 #g of tar per milligram of protein (Fig. 1). The rate of NADH oxidation was approximately twice the rate of oxygen consumption, in agreement with the predicted stoichiometry. When acetonitrile alone was incubated with submitochondrial particles, no effect on the rate of substrate oxidation was observed (data not shown). Similar results with beef heart submitochondrial particles were also found (data not shown). A comparison of the sensitivity of NADH-Q reductase, succinate-Q reductase, and the cytochromes to acetonitrile extracts of cigarette tar was investigated utilizing various inhibitors and electron donors. The addition of 180 ~tg of tar extract per milligram of protein to a submitochondrial preparation containing
368
W . A . PRYOR et al. Table 2. Differential Sensitivity of Liver Submitochondrial Electron Transport Components to Cigarette Smoke Tar Extracts a Rate b Treatment
-Tar
NADH NADH+rot NADH+rot+succ NADH+rot+succ+antia NADH + rot + succ + anti a + asc/TMPD/CN c
107_+ 16_+ 67+ 15_+
Percent Inhibition
+Tar 9 4 8 5
19_+ 3 7+ 1 33_+ 6 5_+ 3
82 -51 --
212 + 32
119 _+ 30
40
a All treatments contained 3 mL of 100 m M sodium phosphate/pH 7.4. The concentrations o f N A D H , rotenone (rot), succinate (succ), antimycin a (anti a), ascorbate (asc), tetramethylphenylenediamine (TMPD), cyanide (CN), and tar were 333 #M, 25 ug/mL, 5 mM, 1.67 #M, 1 mM, 1 mM, 1.67 mM, and 186 #g/mg protein, respectively. The protein concentration was 153 #g/mL of liver SMP. Each rate value represents an average of three replications _+ 1 standard deviation. b Reaction rates are expressed as micromoles o f O 2 consumed per minute per milligram of protein. c The autooxidation o f ascorbate and T M P D was accounted for by subtracting the rate after the addition of KCN from the rate before the addition o f KCN.
159 #g of protein/mL inhibits NADH-dependent respiration by 82% (Table 2). Rotenone blocks electron flow to the NADH-Q reductase complex, and the addition of succinate bypasses this inhibition. The addition of tar extract to this rotenone/succinate system results in a 51% inhibition of the electron transport (Table 2). The addition of antimycin a (anti a) blocks electron flow to the succinate-Q reductase complex,
"2
and tetramethylphenylenediamine (TMPD)/ascorbate (asc) can be used to bypass this inhibition by donating electrons to the cytochromes. Both TMPD and ascorbate autooxidize, and the addition of potassium cyanide, which blocks mitochondrial electron flow to oxygen, was used to determine the residue rate of TMPD/ascorbate autooxidation. Addition of tar extract to this TMPD/ascorbate system inhibits respi-
225
225~ ",
180 ~
13.., b/] 180 ...~.~
0
0 L,
•
135 Z
= NADH
o = 02 •
135"~
9O
9O
~
o
o
~
~ 0
45 [~
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"'~ 0 - ~ _ ~ _ 0
0
i
I
I
I
IO0
200
300
400
Tar Concentration
o 0
500
~
t~
(ug t a r / m g P r o £ )
Fig. 1. Dose-dependent inhibition of both oxygen uptake and N A D H oxidation in submitochondrial particles exposed to cigarette tar extract. Liver submitochondrial particles were treated with increasing amounts o f tar extract; reactions were initiated by the addition of NADH. The total assay volume was 3 m L and contained 100 m M sodium phosphate/pH 7.4 and 333 #M NADH. The data are expressed as nanomoles of oxygen consumed per minute per milligram of submitochondrial particles protein or nanomoles N A D H oxidized per minute per milligram of protein. Values are the average of two determinations. Acetonitrile addition did not alter the rate o f N A D H oxidation: A control without acetonitrile gave 202 _+ 7 nmol NADH oxidized per minute per milligram o f protein, and the addition o f 20 #L aeetonitrile to this experiment resulted in 198 ___5 nmol N A D H oxidized per minute per milligram of protein.
Effect of cigarette tar on electron transport
0.0010 ~__... ~- " ~ . ~ - 0,0792 ~ #-~- .
.
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0891-5849/92 $5.00 + .00 Copyright © 1992 Pergamon Press Ltd.
Original Contribution THE I N H I B I T O R Y
EFFECT OF EXTRACTS OF CIGARETTE TAR ELECTRON TRANSPORT OF MITOCHONDRIA AND SUBMITOCHONDRIAL PARTICLES
ON
WILLIAM A. PRYOR, NANCY C. ARBOUR, BRAD UPHAM, and DANIEL F. C H U R C H Biodynamics Institute, Louisiana State University, Baton Rouge, LA 70803, U.S.A. (Received 17 September 1991; Revised 30 December 1991 ; Accepted 2 January 1992)
Abstract--Acetonitrile extracts of cigarette tar inhibit state 3 and state 4 respiration of intact mitochondria. Exposure of respiring submitochondrial particles to acetonitrile extracts of cigarette tar results in a dose-dependent inhibition of oxygen consumption and reduced nicotinamide adenine dinucleotide (NADH) oxidation. This inhibition was not due to a solvent effect since acetonitrile alone did not alter oxygen consumption or NADH oxidation. Intact mitochondria are less sensitive to extracts of tar than submitochondrial particles. The NADH-ubiquinone (Q) reductase complex is more sensitive to inhibition by tar extract than the succinate-Q reductase and cytochrome complexes. Nicotine or catechol did not inhibit respiration of intact mitochondria. Treatment of submitochondrial particles with cigarette tar results in the formation of hydroxyl radicals, detected by electron spin resonance (ESR) spin trapping. The ESR signal attributable to the hydroxyl radical spin adduct requires the presence of NADH and is completely abolished by catalase and to a lesser extent superoxide dismutase (SOD). Catalase and SOD did not protect the mitochondrial respiratory chain from inhibition by tar extract, indicating that the radicals detected by ESR spin trapping are not responsible for the inhibition of the electron transport. We propose that tar causes at least two effects: ( l ) Tar components interact with the electron transport chain and inhibit electron flow, and (2) tar components interact with the electron transport chain, ultimately to form hydroxyl radicals. Keywords--Mitochondria, Cigarette tar, Electron transport, Autooxidation, Electron spin resonance, Spin trapping, Free radicals
mitochondria. Consistent with this hypothesis, exposure to cigarette tar has been shown to alter mitochondrial ultrastructure in cultured protozoa) ° Several studies have indicated that cigarette smoke inhibits mammalian mitochondrial function. 11:2 In one of the earliest reports, Kolberg x3 suggested that the depletion of oxygen by lung tissue homogenates was caused by cigarette-smoke-induced damage to mitochondrial membranes. Kyle et al. 14 demonstrated decreased phosphorylative efficiency in guinea pig lung mitochondria exposed to whole tobacco smoke in vivo. Several in vitro studies by Gairola et al. 15-17 showed that whole smoke alters respiration from pyridine-linked or ravin-linked substrates in intact mitochondria; depending on the protocol for the preparation of the tobacco smoke extract, either partial stimulation or inhibition of oxygen utilization by the mitochondria was observed. Attempts were made to localize the electron transport chain component(s) with which cigarette smoke interacted; spectroscopic measurement of the cytochromes in their reduced
INTRODUCTION
Over 4000 compounds, many of them highly toxic, have been identified in tobacco smoke.~-~ There is epidemiological evidence linking cigarette smoke with cancers of the larynx and lung, 4'5 and smoking has been suggested to be responsible for some or most of the deaths due to emphysema and chronic obstructive lung disease. 6,7Cigarette tar has been shown to possess redox properties that alter the activity of biologically important macromolecules. For example, Bilimoria et al. 8 have reported that tar accelerates the oxidation of ascorbate by a free-radical-generating reaction. Another study by these workers has shown that cigarette tar reduces cytochrome c,9 suggesting that tar might interact with and affect the electron transport chain of
Address correspondence to William A. Pryor and Daniel F. Church, Biodynamics Institute, Louisiana State University, Baton Rouge, LA 70803. 365
366
W.A. PRYOR et al.
state indicated that multiple cytochrome constituents are involved in the interaction of whole cigarette smoke and intact mitochondria. ~6 In the present study, we report an investigation of the mechanisms involved in cigarette-tar-mediated inhibition of mitochondrial electron transport and compare the effects in mitochondria isolated from beef heart and liver. We have incubated acetonitrile extracts of cigarette tar with intact mitochondria and submitochondrial particles (SMP) and followed both oxygen consumption and reduced nicotinamide adenine dinucleotide (NADH) oxidation. We also performed electron spin resonance (ESR) spin-trapping studies to ascertain whether oxy radicals are involved in the interaction of cigarette tar with SMP. MATERIALS AND M E T H O D S
Materials Research cigarettes (1RI series) were obtained from the Tobacco & Health Research Institute, University of Kentucky (Lexington, Kentucky). The nitrone spin trap, 5,5-dimethyl-l-pyrroline-N-oxide (DMPO), was purchased from Sigma Chemical Co. (St. Louis, MO) and purified with activated charcoal as described by Buettner and Oberley. 18 All other chemicals were used as supplied. Deionized water was used to prepare reagents. Buffers were treated with Chelex-100 to remove any contaminating iron. All reagents were prepared in water, except rotenone and antimycin, which were dissolved in ethanol and methanol, respectively.
Preparation of beef heart submitochondrial particles Fresh bovine hearts were obtained from a local slaughterhouse and were kept at 4°C throughout the procedure. Beef heart was minced into 1 in 3 pieces, and 200 g of this tissue were homogenized using a Waring blender in 0.25 M sucrose, 0.01 M Tris, pH 7.8. The pH of the resulting homogenate was adjusted to 7.8 with 6 M KOH. Mitochondria were isolated from the homogenate as described by Smith. 19 The mitochondria were resuspended in 250 mM sucrose, 1 mM succinate, 2 mM ethylenediaminetetraacetic acid (EDTA), l0 mM Tris, pH 7.8. After a 1-week storage at -20°C, the mitochondria were thawed and recentrifuged. The pellets were then homogenized in 100 mM sodium phosphate, pH 7.8. Submitochondrial particles were prepared in phosphate buffer according to Davies et al. 2° The Bradford method of protein determination was performed on the fresh preparation using bovine serum albumin as a stan-
dard. 21 Aliquots of the submitochondrial suspension were stored up to three months at -80°C.
Preparation of rat liver mitochondria Sprague-Dawley rats were fasted overnight and sacrificed by decapitation. The liver was removed and macerated with scissors in a buffer containing 210 mM mannitol, 70 mM sucrose, 5 mM HEPES/pH 7.4, 1 mM [ethylenebis-(oxyethylenenitrilo)]tetraacetic acid (EGTA), 1 mM EDTA, and 0.15% lipidfree bovine serum albumin. The macerated tissue was homogenized using a Potter-Elvehjem homogenizer with a teflon pestle. The homogenate was centrifuged at 200 × g for 10 min at 4°C. The supernatant solution was centrifuged at 6000 × g for 10 min at 4°C. The pellet was resuspended in a buffer containing 210 mM mannitol, 70 mM sucrose, and 5 mM HEPES/ pH 7.4. All assays with mitochondria were conducted in the resuspension buffer plus 5 mM MgCI2 and 5 mM potassium phosphate. Liver submitochondrial particles were prepared the same way as beef heart submitochondrial particles.
Cigarette tar preparation Standard conditions of storing and smoking the cigarettes were employed,z2,23 Prior to use, the cigarettes were conditioned at room temperature by storage over a saturated solution of ammonium nitrate. The tar from three cigarettes was collected on a Cambridge filter by drawing a 35-mL puffof smoke, 2 s in duration, once a minute through the filter. The filter was then placed in 7.5 mL of a solvent and allowed to soak for 5 min, the extract filtered and kept on ice. The solvents tested include acetonitrile, dimethyl sulfoxide (DMSO), and ethanol. To quantify the amount of cigarette tar collected, the filter was weighed after smoking the cigarettes (wet weight) and then after drying the filter completely (dry weight). On average, a weight corresponding to 22 mg of wet tar per cigarette (66 mg total from three cigarettes) was collected on the Cambridge filter. Quantitative measurements indicated that acetonitrile extracts approximately 40 mg of the 66 mg total wet tar (i.e., it extracts 13 mg/ cigarette). The other solvents tested appear to have an efficiency for extracting tar components that is very similar (see Table 1). The weight of tar described in the table and figure legends is the dry weight. While it cannot be assumed that all three solvents extract the same components from tar with the same efficiency, the tar extracts of all three appear to affect submitochondrial particles respiration similarly (see Table 1). Acetonitrile was used for most of our experiments be-
Effect of cigarette tar on electron transport Table 1. Extraction o f Cigarette Tar Components by Various Solvents: Effect on Oxygen Consumption by Beef Heart Submitochondrial Particles a Reaction Mixture NADH NADH NADH NADH NADH
+ + + +
Acetonitrile Tar-Ethanol Extract Tar-DMSO Extract Tar-Acetonitrile Extract
Rate b 151.0 137.0 23.0 21.0 18.0
+ 7.0 _+ 6.0 _+ 2.0 _ 4.0 _+ 2.0
a All treatments contained 100 m M sodium phosphate/pH 7.4. Concentrations of N A D H and protein arc 1.3 m M and 0.3 mg protein/mL, respectively. 50 uL of acetonitrile or tar extract containing approximately 300, 270, and 280 ug o f dry tar in ethanol, DMSO, and acetonitrile, respectively, was added per reaction mixture. Values are the average of two determinations. b Rate = nanomoles o f O 2 consumed/per minute per milligram of protein.
cause of its volatility and the ease of evaporating it from the Cambridge filters.
Respiration measurement in submitochondrial particles Respiration by submitochondrial particles was measured polarographically at 25°C with a Clarketype electrode (Yellow Springs Inst. Company, Yellow Springs, CO). Mitochondria were incubated in air-saturated medium containing 100 mM sodium phosphate, pH 7.8, giving a final volume of 3 mL. Either 1.3 mM NADH or 13 mM sodium succinate was used to initiate oxygen consumption.
NADH oxidation Oxidation of NADH was measured by following the absorbance decrease at 340 nm, using an extinction coefficient of 6.23 mM -~ cm-'. Absorbance changes were measured with a Hewlett Packard 8415A Diode Array Spectrophotometer. The reaction mixture contained submitochondrial particles (0.7 mg of protein/mL) in 1.0 mL of the medium used in respiration studies. After a 5-min incubation, tar extract was added followed with the addition of NADH.
ESR spectroscopy Spectra were recorded on a Bruker 100D X-band spectrometer equipped with an Aspect 2000 data acquisition system. The reaction mixture containing submitochondrial particles (3 mg/mL protein), 10 mM NADH, and 50 mM DMPO in 100 mM sodium phosphate, pH 7.8, was allowed to incubate for 1 min at room temperature. Tar extract was then added and
367
the sample mixed and purged with nitrogen. When included, scavengers were added prior to the addition of tar. The sample was transferred to a 17-mm quartz flat cell and degassed by vacuum before placing in the ESR cavity. Typically, three to five 200-s scans of each spectrum were recorded with a modulation frequency of 100 kHz, modulation amplitude 1.25 G, microwave power l0 mW, microwave frequency 7.4 kHz, and a scan rate of 30 G/min. RESULTS
Effects of extraction solvents Cigarette tar was collected on a Cambridge filter as described in "Materials and Methods," and several solvents were tested for their efficiency at extracting tar components from the filter. Table 1 presents a comparison of the rates of NADH-stimulated oxygen consumption by SMP from beef heart treated with ethanol, DMSO, and acetonitrile extracts of cigarette tar. The data (expressed as nanomoles of oxygen consumed per minute per milligram of protein) indicate that similar inhibited rates of respiration are observed for submitochondrial particles in the presence of tar extracts in any of these three solvents. The addition of acetonitrile to respiring SMP did not alter oxygen consumption; thus, the observed inhibition by tar extracts is not the result of a nonspecific effect of the solvent. Therefore, we adopted acetonitrile as a solvent for subsequent experiments because of its ease of evaporative removal. The effect of acetonitrile extracts of cigarette tar on NADH-stimulated respiration in rat liver SMP also inhibited respiration to the same extent (Tables 1 and 2), indicating that the source of the SMP made no difference. Exposure of rat liver submitochondrial particles to acetonitrile extracts of cigarette tar results in dose-dependent inhibition of both oxygen consumption and NADH oxidation, with maximal inhibition occurring at approximately 250 #g of tar per milligram of protein (Fig. 1). The rate of NADH oxidation was approximately twice the rate of oxygen consumption, in agreement with the predicted stoichiometry. When acetonitrile alone was incubated with submitochondrial particles, no effect on the rate of substrate oxidation was observed (data not shown). Similar results with beef heart submitochondrial particles were also found (data not shown). A comparison of the sensitivity of NADH-Q reductase, succinate-Q reductase, and the cytochromes to acetonitrile extracts of cigarette tar was investigated utilizing various inhibitors and electron donors. The addition of 180 ~tg of tar extract per milligram of protein to a submitochondrial preparation containing
368
W . A . PRYOR et al. Table 2. Differential Sensitivity of Liver Submitochondrial Electron Transport Components to Cigarette Smoke Tar Extracts a Rate b Treatment
-Tar
NADH NADH+rot NADH+rot+succ NADH+rot+succ+antia NADH + rot + succ + anti a + asc/TMPD/CN c
107_+ 16_+ 67+ 15_+
Percent Inhibition
+Tar 9 4 8 5
19_+ 3 7+ 1 33_+ 6 5_+ 3
82 -51 --
212 + 32
119 _+ 30
40
a All treatments contained 3 mL of 100 m M sodium phosphate/pH 7.4. The concentrations o f N A D H , rotenone (rot), succinate (succ), antimycin a (anti a), ascorbate (asc), tetramethylphenylenediamine (TMPD), cyanide (CN), and tar were 333 #M, 25 ug/mL, 5 mM, 1.67 #M, 1 mM, 1 mM, 1.67 mM, and 186 #g/mg protein, respectively. The protein concentration was 153 #g/mL of liver SMP. Each rate value represents an average of three replications _+ 1 standard deviation. b Reaction rates are expressed as micromoles o f O 2 consumed per minute per milligram of protein. c The autooxidation o f ascorbate and T M P D was accounted for by subtracting the rate after the addition of KCN from the rate before the addition o f KCN.
159 #g of protein/mL inhibits NADH-dependent respiration by 82% (Table 2). Rotenone blocks electron flow to the NADH-Q reductase complex, and the addition of succinate bypasses this inhibition. The addition of tar extract to this rotenone/succinate system results in a 51% inhibition of the electron transport (Table 2). The addition of antimycin a (anti a) blocks electron flow to the succinate-Q reductase complex,
"2
and tetramethylphenylenediamine (TMPD)/ascorbate (asc) can be used to bypass this inhibition by donating electrons to the cytochromes. Both TMPD and ascorbate autooxidize, and the addition of potassium cyanide, which blocks mitochondrial electron flow to oxygen, was used to determine the residue rate of TMPD/ascorbate autooxidation. Addition of tar extract to this TMPD/ascorbate system inhibits respi-
225
225~ ",
180 ~
13.., b/] 180 ...~.~
0
0 L,
•
135 Z
= NADH
o = 02 •
135"~
9O
9O
~
o
o
~
~ 0
45 [~
ID
"'~ 0 - ~ _ ~ _ 0
0
i
I
I
I
IO0
200
300
400
Tar Concentration
o 0
500
~
t~
(ug t a r / m g P r o £ )
Fig. 1. Dose-dependent inhibition of both oxygen uptake and N A D H oxidation in submitochondrial particles exposed to cigarette tar extract. Liver submitochondrial particles were treated with increasing amounts o f tar extract; reactions were initiated by the addition of NADH. The total assay volume was 3 m L and contained 100 m M sodium phosphate/pH 7.4 and 333 #M NADH. The data are expressed as nanomoles of oxygen consumed per minute per milligram of submitochondrial particles protein or nanomoles N A D H oxidized per minute per milligram of protein. Values are the average of two determinations. Acetonitrile addition did not alter the rate o f N A D H oxidation: A control without acetonitrile gave 202 _+ 7 nmol NADH oxidized per minute per milligram o f protein, and the addition o f 20 #L aeetonitrile to this experiment resulted in 198 ___5 nmol N A D H oxidized per minute per milligram of protein.
Effect of cigarette tar on electron transport
0.0010 ~__... ~- " ~ . ~ - 0,0792 ~ #-~- .
.
369
+ 100 F.I Tor . . .
u ID
-0,1594
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