Effects of Inhibition and Induction of Cytochrome P-450 Isozymes on Hyperoxic Lung Injury in Rats Nevins W. Todd, Christine M. Hunt, Thomas P. Kennedy, Claude A. Piantadosi, and A. Richard Whorton Departments of Medicine and Pharmacology, Duke University Medical Center, Durham, North Carolina
Pulmonary oxygen toxicity most likely results from excessive production of reactive oxygen species. The role of the cytochromes P-450 in this process is controversial because these enzymes have been reported both to enhance hyperoxic lung injury and to protect from the damaging effects of 100% oxygen. We sought to further determine the role of the cytochromes P-450 in hyperoxic lung injury by inhibiting and inducing pulmonary cytochrome P-450 isozymes in rats. Treatment with the cytochrome P-450 inhibitor cimetidine or 8-methoxypsoralen did not improve survival or reduce lung edema in rats exposed to 100% oxygen. The activity of cytochrome P-450IIB1~ the major pulmonary cytochrome P-450 isozyme in rats, was clearly inhibited by 8-methoxypsoralen. is-Naphthoflavone ({jNF), a selective inducer of cytochrome P-450IA1~ was administered in two-dose and five-dose regimens. The two-dose regimen produced significant and sustained induction of cytochrome P-450IAI activity, but survival in these rats was not improved when exposed to 100% oxygen. In rats treated with five doses of {jNF~ a small increase in survival time was found from 71.1 ± 8.7 to 88.0 ± 20.2 h; however, there was no difference in the induction of cytochrome P-450IAI activity between this five-dose regimen and the two-dose regimen. The small improvement in survival after five doses of {jNF is thus unrelated to cytochrome P-450IAI induction. We conclude that neither inhibition of cytochrome P-450IIBI activity nor induction of cytochrome P-450IAI activity protects adult rats against hyperoxic lung injury.
Hyperoxic lung injury most likely results from the overwhelming production of toxic oxygen species (1). This hypothesis is supported by data that have shown that superoxide anion (Or) and hydrogen peroxide (H20 2 ) are produced during hyperoxia by lung homogenates, mitochondria, and microsomes (2~ 3). The role of the pulmonary cytochromes P-450 in this process is controversial, and their postulated effects have ranged from enhancing hyperoxic lung injury (4) to protecting from the damaging effects of 100% oxygen (5). The cytochromes P-450 are a group of hemoproteins that primarily act as monooxygenases in the synthesis and metabolism of many endogenous and exogenous compounds. A nomenclature system for this group of enzymes has recently been implemented which is based on DNA sequencing of the multiple forms or isozymes of cytochrome P-450 (6). At the present time, four cytochrome P-450 isozymes have been
(Received in original form October 11, 1991 and in revised form February 11, 1992) Address correspondence to: Nevins W. Todd, M.D., Department of Medicine, Box 3315, Duke University Medical Center, Durham, NC 27710.
cn;
Abbreviations: 3-methylcholanthrene, 3MC; hydrogen peroxide, H2 l3-naphthoflavone, I3NF; superoxide anion, Oi; phosphate-buffered saline, PBS. Am. J. Respir. Cell Mol. BioI. \bl. 7. pp. 222-229, 1992
identified in the rat lung: P-450IA1, P-450IIB1, P-450IIE1, and P-450 L-2 (4, 7~ 8). These isozymes can be distinguished from each other by immunologic and biochemical techniques. Pulmonary cytochromes P-450 may potentially enhance hyperoxic lung injury by producing reactive oxygen species. Though the cytochromes P-450 primarily act as monooxygenases, some P-450 isozymes can also function as oxidases and generate H20 2 by the two electron reduction of oxygen (9): O2
+
NADPH
+
H+ - H 20 2
+
NADP+
Purified cytochrome P-450 isozymes as well as lung microsomes have been shown to produce 02 and H20 2 in vitro (2 ~ 10), and several investigators have proposed that the cytochromes P-450 may be an important intracellular source of O; and H 20 2 during hyperoxia. Tindberg and IngelmanSundberg found that cytochrome P-450IIEI protein was increased in lung microsomes of rats exposed to 100% oxygen (4). In addition, induction of cytochrome P-450llEI protein with acetone decreased the survival time of rats exposed to 100 % oxygen (4). The investigators postulated that reactive oxygen species may have been generated during hyperoxia from the high oxidase activity of this cytochrome P-450 isozyme. Gonder and co-workers studied strains of mice that were susceptible to cytochrome P-450 induction by aromatic
Todd, Hunt, Kennedy et al.: Role of Cytochromes P-450 in Hyperoxic Lung Injury
hydrocarbons (11). They observed that hyperoxia also induced cytochrome P-450 protein in these susceptible strains and that these mice died earlier in 100 % oxygen than mice in which the cytochromes P-450 could not be induced. Recently, Hazinski and associates found that treatment with the cytochrome P-450 inhibitor cimetidine significantly reduced lung injury in lambs exposed to 100% oxygen (12). They proposed that inhibition of the cytochromes P-450 with cimetidine reduced the total oxidant stress generated during hyperoxia. Alternatively, the cytochromes P-450 may be able to play a protective role during exposure to high concentrations of oxygen. Some cytochrome P-450 isozymes can utilize organic hydroperoxides to metabolize substrates by a peroxidase-like mechanism as follows (13):
RH + XOOH
-+
ROH
+
XOH
An increase in the peroxidase activity of the cytochromes P-450 could potentially detoxify H202 and other toxic hydroperoxides generated during hyperoxia. Mansour and colleagues have reported that treatment with {3-naphthoflavone ({3NF)or 3-methylcholanthrene (3MC), inducers of cytochrome P-450IAl, significantly prolongs survival in rats (5) and mice (14) exposed to high concentrations of oxygen. Peroxidase activity in lung microsomes from these rats was increased when compared with controls. These investigators stated that induction of the cytochromes P-450 by {3NF -and 3MC may have been responsible for these protective effects. We used a rat model of pulmonary oxygen toxicity to further investigate the ability of the cytochromes P-450 to modulate hyperoxic lung injury. We used cimetidine and 8-methoxypsoralen, two established cytochrome P-450 inhibitors, to evaluate the effects of inhibition of P-450 isozymes on survival and lung edema in rats exposed to 100% oxygen. We also treated rats with {3NF, a selective inducer of cytochrome P-450IAl, to examine the effects of cytochrome P-450IAI induction on pulmonary oxygen toxicity. By quantitating the inhibition and induction of pulmonary cytochrome P-450 isozymes in the rat and relating it to survival and lung edema, we sought to better define the role of the cytochromes P-450 in hyperoxic lung injury.
Materials and Methods Animals Male, virus-free, Sprague-Dawley rats (Charles River, Inc., Wilmington, MA), 275 to 350 g, were used for all experiments and were given free access to food (Purina rat chow) and water. Chemicals Cimetidine (Tagamet) was purchased from SK&F Lab Co. (Cidra, PR). Benzphetamine, ethoxyresorufin, resorufin, 8-methoxypsoralen, and {3NF were purchased from Sigma Chemical Co. (St. Louis, MO). Pentobarbital (Nembutal) was purchased from Abbott Laboratories (North Chicago, IL). Treatment All injections were administered intraperitoneally. Cimetidine was dissolved in saline, and 8-methoxypsoralen was dissolved in olive oil or com oil. For oxygen exposures, cimeti-
223
dine was administered twice daily at a dose of 120 mg/kg, and 8-methoxypsoralen was administered once daily at 27 or 50 mg/kg. The first dose of cimetidine or 8-methoxypsoralen was administered before initiation of oxygen exposure. For the determination of pentobarbital sleeping time and benzphetamine demethylase activity, cimetidine or 8-methoxypsoralen was given as a single dose of 120 or 27 mg/kg, respectively. {3NF was dissolved in olive oil and administered in two different dosing regimens. A two-dose regimen was given in which rats received {3NF (80 mg/kg/dose) 48 h and 1 h before initiation of oxygen exposure. These rats received no further {3NF during the exposure period. A fivedose regimen was administered according to the methods of Mansour and colleagues (5), in which rats received {3NF (70 mg/kg/dose) once daily for 4 days before oxygen exposure and then a fifth dose 36 h after the initiation of oxygen exposure. For all experiments, control animals were treated identically with vehicle, either saline, olive oil, or corn oil. During the oxygen exposures, air-exposed animals were also treated concurrently with cimetidine, 8-methoxypsoralen, ,{3NF, or vehicle. Oxygen Exposure Rats were exposed to 100 % oxygen in plexiglass chambers at a flow rate of 10 liters/min. Oxygen exposure was continued even when the animals were removed from the chambers for treatments. Oxygen percentage was monitored by a polarographic electrode and maintained continuously above 98 %. Temperature was maintained between 20 and 22 0 C, and carbon dioxide concentration was kept below 0.5 %. Survival times in 100% oxygen were determined. Pleural fluid volume was measured by aspirating pleural fluid from the chest cavity through a small incision in the diaphragm. Lung wet/dry weight ratios were calculated from the left lung after drying the tissue for 5 to 7 days at 60 0 C. Microsome Preparation Microsomes were prepared from oxygen- and air-exposed animals. Animals were killed by CO 2 narcosis, and the lungs were removed. Tissue was homogenized with a teflonglass homogenizer using a ~crosomal isolation buffer consisting of 0.1 M sodiunrphosphate (NaH2P04 ) , 2 mM EDTA, pH 7.4. The homogenate was centrifuged at 17,800 x g, and the mitochondrial pellet was discarded. The supernatant was centrifuged at 100,000 x g for 60 min. The microsomal pellet was resuspended in isolation buffer and centrifuged at 100,000 X g for 50 min. Lung microsomes were suspended in microsomal storage buffer (0.1 M potassium phosphate [K2HP04] , 1.0 mM EDTA, 20% glycerol, 1.0 mM dithiothreitol, 20 ~M butylated hydroxytoluene, pH 7.25) and stored at -70 0 C. Microsomal protein content was determined according to a modified method of Lowry (15). Substrate Activities The N-demethylation of benzphetamine (0.5 ~M) was determined according to the method of Furuya and co-workers (16). The O-deethylation of ethoxyresorufin (10 ~M) was determined according to a method of Burke and Mayer (17). Tris-HCI buffer (0.1 M, pH 7.6), ethoxyresorufin, andmicrosomes were added to give a total volume of 3 ml. The reaction was initiated by the addition ofNADPH (0.5 mM), and
224
AMERICAN JOURNAL OF RESPIRATORY CELL AND MOLECULAR BIOLOGY VOL. 7 1992
the formation of resorufin was measured by continuous fluorimetry. The reaction was conducted at 37 0 C. Immunoblot Analysis Microsomal protein was separated by electrophoresis in 1.5 rom X 10 em sodium dodecyl sulfate polyacrylamide (10%) slab gels and then electrophoretically transferred to nitrocellulose sheets. The nitrocellulose sheets were incubated overnight at room temperature in phosphate-buffered saline (PBS) containing 10% dialyzed calf serum and 3 % bovine serum albumin. The sheets were then treated, in order (with intervening PBS washes), with polyclonal monospecific anti3MC antibody (18) (1:500 dilution, donated by Dr. Philip S. Guzelian, Medical College of Virginia, Richmond, VA), peroxidase-conjugated secondary anti-goat antibody, and a solution containing 0.05 M Tris- HCI buffer (pH 7.6) with 0.55 mM 3,3'-diaminobenzidine tetrahydrochloride and 2 mM HzO z. Pentobarbital Sleeping Time Rats were treated with a single dose of cimetidine or 8-methoxypsoralen and concurrently given pentobarbital. Sleeping time was defined as the amount of time during which the animal was unable to put itself into an upright position. Statistical Analysis For Table 3, statistical analysis was performed using a t test of independent means (19). For all other data, statistics were performed using a one-way ANOVA (19). The level of significance was defined at P < 0.05.
Results Effects of Inhibitors of the Cytochromes P-450 on Survival and Lung Edema in Rats Exposed to 100% Oxygen The effects of cytochrome P-450 inhibition on survival were evaluated in animals exposed to hyperoxia. Rats were treated with cimetidine or 8-methoxypsoralen and exposed to 100% oxygen (Os) , and control rats were treated with saline or olive oil and also exposed to 100% oxygen (Table 1). As expected, exposure of the control animals tohyperoxia resulted in their deaths within 72 h. No difference in survival was found, however, between Os-exposed rats treated with cimetidine or saline. Similarly, there was no difference in survival between Os-exposed rats treated with 8-methoxypsoralen or olive oil. There were no deaths in air-exposed animals treated with cimetidine, 8-methoxypsoralen, or vehicle. The effects of cytochrome P-450 inhibition on lung edema also were evaluated in rats exposed to 100% oxygen. Rats were treated with cimetidine, 8-methoxypsoralen, or vehicle, and pleural fluid volume and lung wet/dry weight ratios were determined after 60 h of oxygen exposure (Table 2). Animals treated with cimetidine, 8-methoxypsoralen, or vehicle were also exposed to air. Although significant pleural fluid, accumulated in Os-exposed animals, there was no difference in pleural fluid volume between Oz-exposed rats treated with cimetidine or saline, or between Os-exposed rats treated with 8-methoxypsoralen or corn oil. There was no significant pleural fluid accumulation in the air-exposed
TABLE I
Effects of the cytochrome P-450 inhibitors cimetidine and 8-methoxypsoralen on survival in rats exposed to 100% oxygen* Survival Time Treatment
(h)
Cimetidine Control (saline)
64.2 ± 6.9 66.9 ± 5.0
8-Methoxypsoralen Control (olive oil)
65.6 ± 6.5 63.9 ± 4.8
* Rats were treated twice daily with cimetidine (120 mg/kg, n = 13) or once daily with 8-methoxypsoralen (27 mg/kg, n = 8) and exposed to 100% oxygen. Control animals treated with saline (n = 12) or olive oil (n = 8) were also exposed to 100% oxygen. There were no deaths in air-exposed animals treated with cimetidine (n = 8), saline (n = 8), 8-methoxypsoralen (n = 4), or olive oil (n = 4). Data are mean ± SD.
rats. Lung wet/dry weight ratios also showed no difference between Os-exposed rats treated with cimetidine, 8-methoxypsoralen, or vehicle. Effects of Cytochrome P-450 Inhibitors on Benzphetamine Demethylase Activity and Pentobarbital Sleeping Time Since neither cimetidine nor 8-methoxypsoralen improved survival or reduced lung edema in 100% oxygen, we sought to verify that these drugs inhibited the pulmonary cytochromes P-450 by measuring the benzphetamine demethylase activity of lung microsomes and the pentobarbital sleeping time. Cytochrome P-450IIB1 is constitutively present in rat lungs and is the major pulmonary cytochrome P-450 isozyme in rats (7). Cytochrome P-450IIBI exhibits high monooxygenase activity toward the substrate benzphetamine (20), and we used the N-demethylation of this substrate to evaluate inhibition of P-450Iffi1. Benzphetamine demethylase activity of lung microsomes was measured from air-exposed rats treated with a single dose of either cimetidine or 8-methoxypsoralen (Figure 1). Cimetidine is a reversible inhibitor of the cytochromes P-450 (21), whereas 8-methoxypsoralen is
TABLE 2
Effects of cimetidine and 8-methoxypsoralen on pleural fluid volume and lung wet/dry weight ratios in rats exposed to 100% oxygen* Pleural Fluid Volume Treatment
(ml)
± 1.2 ± 1.9
Lung Wet/ Dry Weight Ratio
Oz/cimetidine Oz/control (saline) Air/ cimetidine Air/control (saline)
11.7 11.1
E ......
0.18
.!:
E
...... '0
0.12
E
Pulmonary oxygen toxicity most likely results from excessive production of reactive oxygen species. The role of the cytochromes P-450 in this process is controversial because these enzymes have been reported both to enhance hyperoxic lung injury and to protect from the damaging effects of 100% oxygen. We sought to further determine the role of the cytochromes P-450 in hyperoxic lung injury by inhibiting and inducing pulmonary cytochrome P-450 isozymes in rats. Treatment with the cytochrome P-450 inhibitor cimetidine or 8-methoxypsoralen did not improve survival or reduce lung edema in rats exposed to 100% oxygen. The activity of cytochrome P-450IIB1~ the major pulmonary cytochrome P-450 isozyme in rats, was clearly inhibited by 8-methoxypsoralen. is-Naphthoflavone ({jNF), a selective inducer of cytochrome P-450IA1~ was administered in two-dose and five-dose regimens. The two-dose regimen produced significant and sustained induction of cytochrome P-450IAI activity, but survival in these rats was not improved when exposed to 100% oxygen. In rats treated with five doses of {jNF~ a small increase in survival time was found from 71.1 ± 8.7 to 88.0 ± 20.2 h; however, there was no difference in the induction of cytochrome P-450IAI activity between this five-dose regimen and the two-dose regimen. The small improvement in survival after five doses of {jNF is thus unrelated to cytochrome P-450IAI induction. We conclude that neither inhibition of cytochrome P-450IIBI activity nor induction of cytochrome P-450IAI activity protects adult rats against hyperoxic lung injury.
Hyperoxic lung injury most likely results from the overwhelming production of toxic oxygen species (1). This hypothesis is supported by data that have shown that superoxide anion (Or) and hydrogen peroxide (H20 2 ) are produced during hyperoxia by lung homogenates, mitochondria, and microsomes (2~ 3). The role of the pulmonary cytochromes P-450 in this process is controversial, and their postulated effects have ranged from enhancing hyperoxic lung injury (4) to protecting from the damaging effects of 100% oxygen (5). The cytochromes P-450 are a group of hemoproteins that primarily act as monooxygenases in the synthesis and metabolism of many endogenous and exogenous compounds. A nomenclature system for this group of enzymes has recently been implemented which is based on DNA sequencing of the multiple forms or isozymes of cytochrome P-450 (6). At the present time, four cytochrome P-450 isozymes have been
(Received in original form October 11, 1991 and in revised form February 11, 1992) Address correspondence to: Nevins W. Todd, M.D., Department of Medicine, Box 3315, Duke University Medical Center, Durham, NC 27710.
cn;
Abbreviations: 3-methylcholanthrene, 3MC; hydrogen peroxide, H2 l3-naphthoflavone, I3NF; superoxide anion, Oi; phosphate-buffered saline, PBS. Am. J. Respir. Cell Mol. BioI. \bl. 7. pp. 222-229, 1992
identified in the rat lung: P-450IA1, P-450IIB1, P-450IIE1, and P-450 L-2 (4, 7~ 8). These isozymes can be distinguished from each other by immunologic and biochemical techniques. Pulmonary cytochromes P-450 may potentially enhance hyperoxic lung injury by producing reactive oxygen species. Though the cytochromes P-450 primarily act as monooxygenases, some P-450 isozymes can also function as oxidases and generate H20 2 by the two electron reduction of oxygen (9): O2
+
NADPH
+
H+ - H 20 2
+
NADP+
Purified cytochrome P-450 isozymes as well as lung microsomes have been shown to produce 02 and H20 2 in vitro (2 ~ 10), and several investigators have proposed that the cytochromes P-450 may be an important intracellular source of O; and H 20 2 during hyperoxia. Tindberg and IngelmanSundberg found that cytochrome P-450IIEI protein was increased in lung microsomes of rats exposed to 100% oxygen (4). In addition, induction of cytochrome P-450llEI protein with acetone decreased the survival time of rats exposed to 100 % oxygen (4). The investigators postulated that reactive oxygen species may have been generated during hyperoxia from the high oxidase activity of this cytochrome P-450 isozyme. Gonder and co-workers studied strains of mice that were susceptible to cytochrome P-450 induction by aromatic
Todd, Hunt, Kennedy et al.: Role of Cytochromes P-450 in Hyperoxic Lung Injury
hydrocarbons (11). They observed that hyperoxia also induced cytochrome P-450 protein in these susceptible strains and that these mice died earlier in 100 % oxygen than mice in which the cytochromes P-450 could not be induced. Recently, Hazinski and associates found that treatment with the cytochrome P-450 inhibitor cimetidine significantly reduced lung injury in lambs exposed to 100% oxygen (12). They proposed that inhibition of the cytochromes P-450 with cimetidine reduced the total oxidant stress generated during hyperoxia. Alternatively, the cytochromes P-450 may be able to play a protective role during exposure to high concentrations of oxygen. Some cytochrome P-450 isozymes can utilize organic hydroperoxides to metabolize substrates by a peroxidase-like mechanism as follows (13):
RH + XOOH
-+
ROH
+
XOH
An increase in the peroxidase activity of the cytochromes P-450 could potentially detoxify H202 and other toxic hydroperoxides generated during hyperoxia. Mansour and colleagues have reported that treatment with {3-naphthoflavone ({3NF)or 3-methylcholanthrene (3MC), inducers of cytochrome P-450IAl, significantly prolongs survival in rats (5) and mice (14) exposed to high concentrations of oxygen. Peroxidase activity in lung microsomes from these rats was increased when compared with controls. These investigators stated that induction of the cytochromes P-450 by {3NF -and 3MC may have been responsible for these protective effects. We used a rat model of pulmonary oxygen toxicity to further investigate the ability of the cytochromes P-450 to modulate hyperoxic lung injury. We used cimetidine and 8-methoxypsoralen, two established cytochrome P-450 inhibitors, to evaluate the effects of inhibition of P-450 isozymes on survival and lung edema in rats exposed to 100% oxygen. We also treated rats with {3NF, a selective inducer of cytochrome P-450IAl, to examine the effects of cytochrome P-450IAI induction on pulmonary oxygen toxicity. By quantitating the inhibition and induction of pulmonary cytochrome P-450 isozymes in the rat and relating it to survival and lung edema, we sought to better define the role of the cytochromes P-450 in hyperoxic lung injury.
Materials and Methods Animals Male, virus-free, Sprague-Dawley rats (Charles River, Inc., Wilmington, MA), 275 to 350 g, were used for all experiments and were given free access to food (Purina rat chow) and water. Chemicals Cimetidine (Tagamet) was purchased from SK&F Lab Co. (Cidra, PR). Benzphetamine, ethoxyresorufin, resorufin, 8-methoxypsoralen, and {3NF were purchased from Sigma Chemical Co. (St. Louis, MO). Pentobarbital (Nembutal) was purchased from Abbott Laboratories (North Chicago, IL). Treatment All injections were administered intraperitoneally. Cimetidine was dissolved in saline, and 8-methoxypsoralen was dissolved in olive oil or com oil. For oxygen exposures, cimeti-
223
dine was administered twice daily at a dose of 120 mg/kg, and 8-methoxypsoralen was administered once daily at 27 or 50 mg/kg. The first dose of cimetidine or 8-methoxypsoralen was administered before initiation of oxygen exposure. For the determination of pentobarbital sleeping time and benzphetamine demethylase activity, cimetidine or 8-methoxypsoralen was given as a single dose of 120 or 27 mg/kg, respectively. {3NF was dissolved in olive oil and administered in two different dosing regimens. A two-dose regimen was given in which rats received {3NF (80 mg/kg/dose) 48 h and 1 h before initiation of oxygen exposure. These rats received no further {3NF during the exposure period. A fivedose regimen was administered according to the methods of Mansour and colleagues (5), in which rats received {3NF (70 mg/kg/dose) once daily for 4 days before oxygen exposure and then a fifth dose 36 h after the initiation of oxygen exposure. For all experiments, control animals were treated identically with vehicle, either saline, olive oil, or corn oil. During the oxygen exposures, air-exposed animals were also treated concurrently with cimetidine, 8-methoxypsoralen, ,{3NF, or vehicle. Oxygen Exposure Rats were exposed to 100 % oxygen in plexiglass chambers at a flow rate of 10 liters/min. Oxygen exposure was continued even when the animals were removed from the chambers for treatments. Oxygen percentage was monitored by a polarographic electrode and maintained continuously above 98 %. Temperature was maintained between 20 and 22 0 C, and carbon dioxide concentration was kept below 0.5 %. Survival times in 100% oxygen were determined. Pleural fluid volume was measured by aspirating pleural fluid from the chest cavity through a small incision in the diaphragm. Lung wet/dry weight ratios were calculated from the left lung after drying the tissue for 5 to 7 days at 60 0 C. Microsome Preparation Microsomes were prepared from oxygen- and air-exposed animals. Animals were killed by CO 2 narcosis, and the lungs were removed. Tissue was homogenized with a teflonglass homogenizer using a ~crosomal isolation buffer consisting of 0.1 M sodiunrphosphate (NaH2P04 ) , 2 mM EDTA, pH 7.4. The homogenate was centrifuged at 17,800 x g, and the mitochondrial pellet was discarded. The supernatant was centrifuged at 100,000 x g for 60 min. The microsomal pellet was resuspended in isolation buffer and centrifuged at 100,000 X g for 50 min. Lung microsomes were suspended in microsomal storage buffer (0.1 M potassium phosphate [K2HP04] , 1.0 mM EDTA, 20% glycerol, 1.0 mM dithiothreitol, 20 ~M butylated hydroxytoluene, pH 7.25) and stored at -70 0 C. Microsomal protein content was determined according to a modified method of Lowry (15). Substrate Activities The N-demethylation of benzphetamine (0.5 ~M) was determined according to the method of Furuya and co-workers (16). The O-deethylation of ethoxyresorufin (10 ~M) was determined according to a method of Burke and Mayer (17). Tris-HCI buffer (0.1 M, pH 7.6), ethoxyresorufin, andmicrosomes were added to give a total volume of 3 ml. The reaction was initiated by the addition ofNADPH (0.5 mM), and
224
AMERICAN JOURNAL OF RESPIRATORY CELL AND MOLECULAR BIOLOGY VOL. 7 1992
the formation of resorufin was measured by continuous fluorimetry. The reaction was conducted at 37 0 C. Immunoblot Analysis Microsomal protein was separated by electrophoresis in 1.5 rom X 10 em sodium dodecyl sulfate polyacrylamide (10%) slab gels and then electrophoretically transferred to nitrocellulose sheets. The nitrocellulose sheets were incubated overnight at room temperature in phosphate-buffered saline (PBS) containing 10% dialyzed calf serum and 3 % bovine serum albumin. The sheets were then treated, in order (with intervening PBS washes), with polyclonal monospecific anti3MC antibody (18) (1:500 dilution, donated by Dr. Philip S. Guzelian, Medical College of Virginia, Richmond, VA), peroxidase-conjugated secondary anti-goat antibody, and a solution containing 0.05 M Tris- HCI buffer (pH 7.6) with 0.55 mM 3,3'-diaminobenzidine tetrahydrochloride and 2 mM HzO z. Pentobarbital Sleeping Time Rats were treated with a single dose of cimetidine or 8-methoxypsoralen and concurrently given pentobarbital. Sleeping time was defined as the amount of time during which the animal was unable to put itself into an upright position. Statistical Analysis For Table 3, statistical analysis was performed using a t test of independent means (19). For all other data, statistics were performed using a one-way ANOVA (19). The level of significance was defined at P < 0.05.
Results Effects of Inhibitors of the Cytochromes P-450 on Survival and Lung Edema in Rats Exposed to 100% Oxygen The effects of cytochrome P-450 inhibition on survival were evaluated in animals exposed to hyperoxia. Rats were treated with cimetidine or 8-methoxypsoralen and exposed to 100% oxygen (Os) , and control rats were treated with saline or olive oil and also exposed to 100% oxygen (Table 1). As expected, exposure of the control animals tohyperoxia resulted in their deaths within 72 h. No difference in survival was found, however, between Os-exposed rats treated with cimetidine or saline. Similarly, there was no difference in survival between Os-exposed rats treated with 8-methoxypsoralen or olive oil. There were no deaths in air-exposed animals treated with cimetidine, 8-methoxypsoralen, or vehicle. The effects of cytochrome P-450 inhibition on lung edema also were evaluated in rats exposed to 100% oxygen. Rats were treated with cimetidine, 8-methoxypsoralen, or vehicle, and pleural fluid volume and lung wet/dry weight ratios were determined after 60 h of oxygen exposure (Table 2). Animals treated with cimetidine, 8-methoxypsoralen, or vehicle were also exposed to air. Although significant pleural fluid, accumulated in Os-exposed animals, there was no difference in pleural fluid volume between Oz-exposed rats treated with cimetidine or saline, or between Os-exposed rats treated with 8-methoxypsoralen or corn oil. There was no significant pleural fluid accumulation in the air-exposed
TABLE I
Effects of the cytochrome P-450 inhibitors cimetidine and 8-methoxypsoralen on survival in rats exposed to 100% oxygen* Survival Time Treatment
(h)
Cimetidine Control (saline)
64.2 ± 6.9 66.9 ± 5.0
8-Methoxypsoralen Control (olive oil)
65.6 ± 6.5 63.9 ± 4.8
* Rats were treated twice daily with cimetidine (120 mg/kg, n = 13) or once daily with 8-methoxypsoralen (27 mg/kg, n = 8) and exposed to 100% oxygen. Control animals treated with saline (n = 12) or olive oil (n = 8) were also exposed to 100% oxygen. There were no deaths in air-exposed animals treated with cimetidine (n = 8), saline (n = 8), 8-methoxypsoralen (n = 4), or olive oil (n = 4). Data are mean ± SD.
rats. Lung wet/dry weight ratios also showed no difference between Os-exposed rats treated with cimetidine, 8-methoxypsoralen, or vehicle. Effects of Cytochrome P-450 Inhibitors on Benzphetamine Demethylase Activity and Pentobarbital Sleeping Time Since neither cimetidine nor 8-methoxypsoralen improved survival or reduced lung edema in 100% oxygen, we sought to verify that these drugs inhibited the pulmonary cytochromes P-450 by measuring the benzphetamine demethylase activity of lung microsomes and the pentobarbital sleeping time. Cytochrome P-450IIB1 is constitutively present in rat lungs and is the major pulmonary cytochrome P-450 isozyme in rats (7). Cytochrome P-450IIBI exhibits high monooxygenase activity toward the substrate benzphetamine (20), and we used the N-demethylation of this substrate to evaluate inhibition of P-450Iffi1. Benzphetamine demethylase activity of lung microsomes was measured from air-exposed rats treated with a single dose of either cimetidine or 8-methoxypsoralen (Figure 1). Cimetidine is a reversible inhibitor of the cytochromes P-450 (21), whereas 8-methoxypsoralen is
TABLE 2
Effects of cimetidine and 8-methoxypsoralen on pleural fluid volume and lung wet/dry weight ratios in rats exposed to 100% oxygen* Pleural Fluid Volume Treatment
(ml)
± 1.2 ± 1.9
Lung Wet/ Dry Weight Ratio
Oz/cimetidine Oz/control (saline) Air/ cimetidine Air/control (saline)
11.7 11.1
E ......
0.18
.!:
E
...... '0
0.12
E
