Vol. 185, No. 3, 1992
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
June 30, 1992
Pages
1083-l
090
SUPPRESSIVE EFFECT OF INTERLEUKIN-1 ON PULMONARY CYTOCHROME I’450 AND SUPEROXIDE ANION PRODUCTION Hironori
Department
Received
Sakai, Toshihiro Okamoto, Reiko Yamamoto, Ram K. Sindhu and Yutaka Kikkawa
of Pathology,
May 18,
College of Medicine, University Irvine, California 92717
of California,
Irvine
199'2
SUMMARY Interleukin-1 has been shown to prolong the survival of rats exposed to lethal concentrations of oxygen. This oxygen tolerance has been attributed by some workers to an increase of manganese superoxide dismutase. We report here that the administration of interleukin-1 to male adult rats results in (i) significant decrease of pulmonary cytochrome P450 at 24 and 72 hours, (ii) decrease of I’450 IIBl mRNA at 24 and 72 hours and (iii) significant decrease of superoxide anion generation from pulmonary microsomes isolated from treated rats. To the best of our knowledge, this is the first report to demonstrate these effects of interleukin-1 on pulmonary P450 and its oxidase activity (O-2 generation). On the basis of these results and several earlier reports in which various P450 depressants have been shown to depress superoxide production from microsomes and to prolong the lives of rodents in hyperoxia, we conclude that oxygen tolerance induced by interleukin-1 administration is likewise mediated, at least in part, by reduced generation of 0 1992Academic superoxide anion from cytochrome I’450 monooxygenase system. PIfzS5,Inc. Oxidant stresses result in the formation of reactive oxygen metabolites (ROS): superoxide anion, hydrogen peroxide, hydroxyl radical, singlet oxygen and other peroxides. The effects of these ROS are neutralized by endogenous antioxidants such as superoxide dismutase (SOD), glutathione, glutathione peroxidase, catalase, vitamin E, etc. Thus, it is evident that the damaging effects of oxygen is dependent upon the balance between ROS and the antioxidant defense system (1). Interleukin-1 (IL-l) and tumor necrosis factor (TNF) when administered i.v. and i.p. prolong the survival of rats in continuous hyperoxia (2,3). Likewise, endotracheal insufflation of IL-1 has been shown to increase the survival of rats in hyperoxia (4). These authors have suggested that prolonged survival of animals after IL-l administration is due to an increase of SOD activity occurring after 2.5 to 3 days of hyperoxia. Studies carried out in this laboratory have shown that interferon-inducers, which are known depressants of cytochrome P450, are all associated with the increased survival of rats in hyperoxia (5) and that microsomes generated lesser 0006-291X192
1083
$4.00
Copyright 0 1992 by Academic Press, Inc. All rights of reproduction in any form reserved.
Vol.
185, No. 3, 1992
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
quantities of superoxide anion as early as 3 hours after the administration of interferon-inducers (6). These studies were followed by several reports suggesting the importance of the role of pulmonary cytochrome P450 in hyperoxia. It has been shown that mice with higher level of pulmonary P450 die earlier than those with lower amounts of P450 in hyperoxia (7). Furthermore, lambs treated with cimetidine, an inhibitor of P450, retain many normal pulmonary functions as compared to untreated controls in hyperoxia (8). In most of these studies, there was no change in SOD activities. IL-1 has been shown to decrease cytochrome P450 of rat liver (9), while its effect on pulmonary cytochrome P450 and O-2 generation by its oxidase activity are not known. In this communication, we present results on the changes in pulmonary microsomal monooxygenase enzymes, the alterations of I’450 isozymes and the alterations in superoxide generating capacity of the pulmonary microsomes after the treatment with IL-l. MATERIALS
AND METHODS
Male Sprague-Dawley CD strain viral antigen and pathogen free rats (body weight 290-320g) were obtained from Harlan-Sprague-Dawley (Indianapolis, IN). They were allowed food (rodent laboratory chow) and water ad libitum and kept on a 12-hr light-dark cycle throughout the experiment. After a preliminary dose-effect study, 5 pg of recombinant human IL-1 in a sterile 0.9% saline solution was injected intraperitoneally. Control rats received saline only and had excess to the same amount of food as the experimental group. Twenty four or seventy two hours after treatment with IL-l or saline, rats were anesthetized with sodium pentobarbital. The lungs and livers were perfused with 0.9% NaCl containing 1 mM EDTA. The lung tissues were pooled from 2 rats of same the same group, weighed and microsomal fractions were obtained by differential centrifugation, as described before, and stored at -85°C (10). Cytochrome P450 content was determined by CO difference spectra using a molar extinction coefficient of 91 cm-1 mM-1 (11). Cytochrome b5 levels were determined with NADH as the reducing agent using a molar extinction coefficient of 21 cm-1 mM-1 for 556/575 nm (12). NADPH-cytochrome P450 reductase (P450 reductase) was determined by the method of Dignam et aZ. (13) and NADHcytochrome bg reductase (b5 reductase) activity was measured as described by Takesue et al. (14) using potassium ferricyanide as the electron acceptor. Protein was determined according to the method of Lowry et al. (15) with bovine serum albumin as a standard. The 0-deethylation of ‘I-ethoxycoumarin (7-ECOD) was determined by measuring the fluorescence of the deethylated products, 7-hydroxycoumarin according to the method described by Rough et al. (16). Benzphetamine Ndemethylase activity was determined by measuring the amount of formaldehyde generated as described by Werringloer (17). Acetylated cytochrome c was used for the measurement of superoxide (O-2) production according to the method described by Kakinuma et al. (18). Cytochrome c in half saturated sodium acetate was acetylated by incubating it with a 100 molar excess of acetic anhydride. The reaction mixture contained microsomes (1OOpg of liver microsomal protein or 5OOpg of lung microsomal protein), 60pM acetylated cytochrome c in 50 mM potassium phosphate buffer, pH 7.4 in a total volume 1.0 ml, The reaction was started by the addition of 0.5 mM NADPH. The reduction of acetylated cytochrome c was monitored 1084
Vol.
185,
No.
3,
1992
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
spectrophotometrically at 550 nm and the molar extinction coefficient of 21 mM10-l was used. The difference in the rates of acetylated cytochrome c reduction measured in the presence or absence of SOD was used as the measure of the amount of O-2 generated (SOD inhibitable acetylated cytochrome c reduction). SDS-polyacrylamide slab gel electrophoresis (10% acrylamide) was performed according to the method described by Laemmli (19), with Bio-Rad Mini Protean II apparatus. Two pg of protein in each sample were applied. Electrophoretic blotting of the protein was performed as described by Towbin et al. (20). The filters were blocked with 3% gelatin in Tris-buffered saline solution and probed with polyclonal antisera against I?450 IIBl. The antigen-antibody complexes were visualized using alkaline phosphatase-conjugated second antibody. For RNA extraction, lungs were removed from the rats immediately without perfusion, frozen by submersion in liquid nitrogen, and stored at -85°C. Total RNA was extracted by using RNAzol (Tel-Test, TX). The amount of RNA was determined from the absorbance at 260 nm using a Gilford Response Spectrophotometer. The oligomer probes used to assess P450 IAl, lA2, IIEl, IIBl and IIB2 mRNAs have the sequences of 5'-TCTGGTGAGCATCCAGGACA-3',5'-GGAAAAGGAACAAGGGTGGC-3', 5'-TCCAGTGACTGAAGGTGTTCCTT-3' 5'-GGTTGGTAGCCGGTGTGA-3' and 5'GGATGGTGGCCTGTGAGA-3', respectively (21, 22,231. 18s ribosomal RNA probe has a sequence of S-CACCTCTAGCGGCGCAATAC-Y (24). The probes were custom-synthesized by Genosys Biotechnologies, Inc. (Woodlands, TX) and were 5’-end labeled with (Y32~) ATP (specific activity, 6000 Ci/m mol; New England Nuclear, MA) and T4polynucleotide kinase (New England Biolabs, MA). The specific activities of the oligomers used for hybridization were typically 2 9x106 cpm/pmol. For northern blot analysis, 20 pg of total RNA were denatured (65”C/lO min/2.2M formaldehyde), subjected to electrophoresis at 46V on 1.2 % agarose/2.2M formaldehyde gels, transferred to nylon filters (Zeta-probe, Bio-Rad, CA) in HETS (Tel-Test) and immobilized by UV irradiation (Stratagene, UV Stratalinker 2400). Prehybridization and hybridization were performed according to Bio-Rad Zeta-Probe Blotting Membranes Instruction Manual except that pH of the perhybridizing solution was adjusted to 7.0 and 10% dextran sulfate was included in both of these solutions. Autoradiography was carried out after exposure to Kodak X-OMAT AR (XAR) film at -85°C for 48 hrs. Densitometric measurement was performed by using Bio-Rad Model 620 Densitometer. Recombinant human IL-1 a (lot 117-271) was generously provided by Drs. Alvin Stern, Swapan Roy and Peter Lomedico of Hoffmann-La Roche, Nutley, NJ. This lot of IL-1 had a specific activity of 3 x 108 units/mg protein as measured by the DlO assay and contained small amounts of endotoxin (0.74 EU/mg protein). NADPH, NADH, cytochrome c, superoxide dismutase, 7-hydroxycoumarin and benzphetamine were purchased from Sigma Chemical (St. Louis, MO). 7ethoxycoumarin was purchased from Pierce (Rockford, IL). All other chemicals were of analytical grade. Statistical analysis was performed on a Macintosh II with the use of Stat View software. Statistical significance of the difference between concurrent control and treated groups was determined by one-way analysis of variance and the Dunnet t test. RESULTS AND DISCUSSION After IL-1 administration P450 was decreased to 54% (34.8/64.7) of the control value at 24 hours and recovered to 64% (36.6/57.4) at 72 hours when results were expressed on per mg protein basis. Cytochrome bg showed significant decrease to 73% (68.5/44.0) at 24 hours and recovered to 92% (69.7/76.1) at 72 hours. The activity 1085
Vol.
185,
No.
3,
Table
1992
1. Effect
BIOCHEMICAL
of recombinant
AND
Inledeukln-1
BIOPHYSICAL
treatment
RESEARCH
on lung mlcrosomal
COMMUNICATIONS
enzymes
roteln) -YF=-%iT l/m
) 5 (/lun
(3)
60.3 f18.7
1.17 f0.33
82.9 f14.9
1.62 f0.34
87.6 f6.0
1.71 fO.08
3.06 f0.08
59.6 f2.0
Saline
2411t (3)
64.7 f 8.6
1.38 io. 19
94.0 f 4.3
2.00 10.10
81.0 rt5.0
1.72 f0.04
2.86 f0.13
60.7 f1.3
Interleutdn-1
24ht 13)
34.89 f 7.0
0.72’ tcO.23
68.5’ + 4.4
1.38’ fO.10
84.7 f7.5
1.70 1tO.08
2.91 to.24
58.3 f3.3
Saline
72ht (3)
57.4 f20.5
1.21 50.44
76.1 * 7.7
1.61 f0.27
84.6 f2.2
1.78 f0.08
2.89 fO.ll
60.7 f2.1
Interleuktn-1
72ht (41
36.6 f 9.0
0.84 f0.22
69.7 ill.2
1.62 f0.42
79.6 rt7.3
1.83 f0.21
2.73 f0.20
62.7 f8.7
Treament h) Untreated
control
rotein]
[/lung)
Values are the meatiSD. t Hours after recombinant Interleuktn 1 or saline InJectIon. 8 Cytochrome P450 and cytochrome bs content are expressed as pmol 19 NADPH-cytochrome P45O(c) reductase acttvlty Is expressed as nmol mtcrosomat proletn and pmol of cytochrome c reduced per mm per ~$1 NADH-cytochrome bs(fenicyanlde) reductase actlvlty Is expressed of mlcrosomal protetn or lung. Signlftcantly different from concurrent control group, l P< 0.05
(/mg
I’450 F&.&J&& protein) [/lung)
(/mg
bc, P&.&m proteln) (/lung)
per mg of mlcrosomal protein and nmol per lung. of cytochrome c reduced per mln per mg of lung. as pmol of ferrlcyantde reduced per mm per mg
of NADPH P450 reductase or NADH b5 reductase were unaltered (Table 1). This depression of I’450 in the lungs was more pronounced than the degree of depression seen in the liver after the identical treatment with IL-1 (Table 3). This difference is probably due to selectivity of the action of IL-1 towards different I’450 isozymes and to the difference of the constituitive P450 isozymes between the lung and the liver. In the liver, the major constituitive enzyme is P450 IICll which are unaffected by the action of IL-1 (25). In the lung the major forms are I’450 IVB and P450 IIBl (26) and I?450 IA1 has only been found in trace amounts. The degree of depression of pulmonary I’450 content of up to 50% indicates that both I’450 IIBl and I’450 IVB are likely to be reduced by the action of IL-l. The activity of 7-ECOD and BPND was reduced to about 75% and 60% of control value at both 24 and 72 hours, respectively (data not shown). The activity of 7-ECOD is largely attributed to I’450 IA1 with minor contributions from I’450 IIBl and I’450 IVB (26, 27). Our data suggests that reduced amounts of any of three I’450 isozymes may produce this degree of depression. BPND activity is largely attributed to I’450 IIBl (27) and the decrease in this catalytic activity suggests that I?450 IIBl is decreased although no information is available on the role of I’450 IVB in BPND activity. Figure 1 shows Northern blot analysis of I’450 IIBl mRNA along with the results of 18s ribosomal RNA hybridization. We have used mRNA probes for P450 IAl, IA2, IIB2, IIEl in addition to I?450 IIBl probe. However, we were unsuccessful in obtaining signals for mRNA except I?450 IIBl. I’450 IAl, however, has been demonstrated in the lungs of rats with polymerase chain reaction amplification (24). All these probes, however, were successfully utilized for hepatocytes (25). As 1086
Vol.
185,
No.
3,
BIOCHEMICAL
1992
AND
BIOPHYSICAL
12
control
RESEARCH
COMMUNICATIONS
48 0-4
2.0 Kb-
P450
IlBl
18s
rRNA
Figure 1. Northern blot analysis of rat pulmonary P450 IIBl after treatment with IL-l. Twenty lg of total pulmonary RNA per lane from control and experimental group
were
subjected to northern
migration
of P450 IIBl
blot analysis. The arrow indicates the position of
mRNA.
18s rRNA
oligonucleotide
probe
is used as loading
control. shown,
there was
considerable
after administration density
indicated
of IL-l.
depression
of I’450 IIBl mRNA
Densitometric
that I’450 IIBl
mRNA
analysis
after correction
was reduced
values 12 and 48 hours after IL-l administration Figure 2 shows the changes of protein
at 24 and 72 hours for 18s RNA
to 77% and 69% of control
respectively (data not shown). content of P450 IIBl. Densitometric
measurement indicate that there was 20% decrease in protein content at both 24 and 72 hours after IL-l administration (data not shown). These data indicate that IL-l administration content
results
by 24 hours.
monoxygenase
I’450 IIBl
mRNA
These results are consistent
by 12 hours
with
the results
from
microsomes
and protein of preceeding
study.
Superoxide hours after IL-l
in decreased
anion generation administration
was measured
and was expressed per microsomal
at 24 and 72
protein
and per
lung (Table 2). At 24 hours superoxide anion was reduced to 73% (2&O/38.2) of control value (~~0.01) and at 72 hours recovered to 90% (32.8/36.3) of control (p.CO.05). Since liver is the major source of P450, the alterations superoxide
generation
was also studied. Control
on hepatic
I’450 and
Table 3 shows that hepatic cytochrome
24
P450
72 (W
---
cP450
II61
Figure 2. Western blot analysis of pulmonary microsomes from control and IL-l treated rats. Microsomes (2 l.tg protein in each lane) were subjected to SDS polyacrylamide gel (10% gel) electrophoresis followed by electrophoretic transfer to nitrocellulose membranes. The membranes were incubated with monoclonal antibody against P450 IIBl and stained using alkaline phosphatase conjugated second antibody. Each lane represents one rat. 1087
Vol.
BIOCHEMICAL
185, No. 3, 1992
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
Table 2. Effect of recombinant interleukin-1 treatment on lung microsomal superoxide generating activity Treament (n) Untreated Saline Interleukin Saline
Superoxide generating activW$ (/mg protein) (/lung)
control (31
1.78 kO.07
34.6 k1.7
24ht 131
1.79 to.14
38.2 k2.8
1.39** 20.03
28.0** 53.3
1.72 kO.06
36.3 k3.5
1 24ht (31 72ht (3)
Interleukin
1 72ht 1.44* 32.8 kO.22 rt3.3 (41 Values are the me&SD. t Hours after recombinant interleukin 1 or saline injection. 5 Activity is expressed as nmol per min per mg microsomal protein or lung. Significantly different from concurrent control group, *PC 0.05. **P
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
June 30, 1992
Pages
1083-l
090
SUPPRESSIVE EFFECT OF INTERLEUKIN-1 ON PULMONARY CYTOCHROME I’450 AND SUPEROXIDE ANION PRODUCTION Hironori
Department
Received
Sakai, Toshihiro Okamoto, Reiko Yamamoto, Ram K. Sindhu and Yutaka Kikkawa
of Pathology,
May 18,
College of Medicine, University Irvine, California 92717
of California,
Irvine
199'2
SUMMARY Interleukin-1 has been shown to prolong the survival of rats exposed to lethal concentrations of oxygen. This oxygen tolerance has been attributed by some workers to an increase of manganese superoxide dismutase. We report here that the administration of interleukin-1 to male adult rats results in (i) significant decrease of pulmonary cytochrome P450 at 24 and 72 hours, (ii) decrease of I’450 IIBl mRNA at 24 and 72 hours and (iii) significant decrease of superoxide anion generation from pulmonary microsomes isolated from treated rats. To the best of our knowledge, this is the first report to demonstrate these effects of interleukin-1 on pulmonary P450 and its oxidase activity (O-2 generation). On the basis of these results and several earlier reports in which various P450 depressants have been shown to depress superoxide production from microsomes and to prolong the lives of rodents in hyperoxia, we conclude that oxygen tolerance induced by interleukin-1 administration is likewise mediated, at least in part, by reduced generation of 0 1992Academic superoxide anion from cytochrome I’450 monooxygenase system. PIfzS5,Inc. Oxidant stresses result in the formation of reactive oxygen metabolites (ROS): superoxide anion, hydrogen peroxide, hydroxyl radical, singlet oxygen and other peroxides. The effects of these ROS are neutralized by endogenous antioxidants such as superoxide dismutase (SOD), glutathione, glutathione peroxidase, catalase, vitamin E, etc. Thus, it is evident that the damaging effects of oxygen is dependent upon the balance between ROS and the antioxidant defense system (1). Interleukin-1 (IL-l) and tumor necrosis factor (TNF) when administered i.v. and i.p. prolong the survival of rats in continuous hyperoxia (2,3). Likewise, endotracheal insufflation of IL-1 has been shown to increase the survival of rats in hyperoxia (4). These authors have suggested that prolonged survival of animals after IL-l administration is due to an increase of SOD activity occurring after 2.5 to 3 days of hyperoxia. Studies carried out in this laboratory have shown that interferon-inducers, which are known depressants of cytochrome P450, are all associated with the increased survival of rats in hyperoxia (5) and that microsomes generated lesser 0006-291X192
1083
$4.00
Copyright 0 1992 by Academic Press, Inc. All rights of reproduction in any form reserved.
Vol.
185, No. 3, 1992
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
quantities of superoxide anion as early as 3 hours after the administration of interferon-inducers (6). These studies were followed by several reports suggesting the importance of the role of pulmonary cytochrome P450 in hyperoxia. It has been shown that mice with higher level of pulmonary P450 die earlier than those with lower amounts of P450 in hyperoxia (7). Furthermore, lambs treated with cimetidine, an inhibitor of P450, retain many normal pulmonary functions as compared to untreated controls in hyperoxia (8). In most of these studies, there was no change in SOD activities. IL-1 has been shown to decrease cytochrome P450 of rat liver (9), while its effect on pulmonary cytochrome P450 and O-2 generation by its oxidase activity are not known. In this communication, we present results on the changes in pulmonary microsomal monooxygenase enzymes, the alterations of I’450 isozymes and the alterations in superoxide generating capacity of the pulmonary microsomes after the treatment with IL-l. MATERIALS
AND METHODS
Male Sprague-Dawley CD strain viral antigen and pathogen free rats (body weight 290-320g) were obtained from Harlan-Sprague-Dawley (Indianapolis, IN). They were allowed food (rodent laboratory chow) and water ad libitum and kept on a 12-hr light-dark cycle throughout the experiment. After a preliminary dose-effect study, 5 pg of recombinant human IL-1 in a sterile 0.9% saline solution was injected intraperitoneally. Control rats received saline only and had excess to the same amount of food as the experimental group. Twenty four or seventy two hours after treatment with IL-l or saline, rats were anesthetized with sodium pentobarbital. The lungs and livers were perfused with 0.9% NaCl containing 1 mM EDTA. The lung tissues were pooled from 2 rats of same the same group, weighed and microsomal fractions were obtained by differential centrifugation, as described before, and stored at -85°C (10). Cytochrome P450 content was determined by CO difference spectra using a molar extinction coefficient of 91 cm-1 mM-1 (11). Cytochrome b5 levels were determined with NADH as the reducing agent using a molar extinction coefficient of 21 cm-1 mM-1 for 556/575 nm (12). NADPH-cytochrome P450 reductase (P450 reductase) was determined by the method of Dignam et aZ. (13) and NADHcytochrome bg reductase (b5 reductase) activity was measured as described by Takesue et al. (14) using potassium ferricyanide as the electron acceptor. Protein was determined according to the method of Lowry et al. (15) with bovine serum albumin as a standard. The 0-deethylation of ‘I-ethoxycoumarin (7-ECOD) was determined by measuring the fluorescence of the deethylated products, 7-hydroxycoumarin according to the method described by Rough et al. (16). Benzphetamine Ndemethylase activity was determined by measuring the amount of formaldehyde generated as described by Werringloer (17). Acetylated cytochrome c was used for the measurement of superoxide (O-2) production according to the method described by Kakinuma et al. (18). Cytochrome c in half saturated sodium acetate was acetylated by incubating it with a 100 molar excess of acetic anhydride. The reaction mixture contained microsomes (1OOpg of liver microsomal protein or 5OOpg of lung microsomal protein), 60pM acetylated cytochrome c in 50 mM potassium phosphate buffer, pH 7.4 in a total volume 1.0 ml, The reaction was started by the addition of 0.5 mM NADPH. The reduction of acetylated cytochrome c was monitored 1084
Vol.
185,
No.
3,
1992
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
spectrophotometrically at 550 nm and the molar extinction coefficient of 21 mM10-l was used. The difference in the rates of acetylated cytochrome c reduction measured in the presence or absence of SOD was used as the measure of the amount of O-2 generated (SOD inhibitable acetylated cytochrome c reduction). SDS-polyacrylamide slab gel electrophoresis (10% acrylamide) was performed according to the method described by Laemmli (19), with Bio-Rad Mini Protean II apparatus. Two pg of protein in each sample were applied. Electrophoretic blotting of the protein was performed as described by Towbin et al. (20). The filters were blocked with 3% gelatin in Tris-buffered saline solution and probed with polyclonal antisera against I?450 IIBl. The antigen-antibody complexes were visualized using alkaline phosphatase-conjugated second antibody. For RNA extraction, lungs were removed from the rats immediately without perfusion, frozen by submersion in liquid nitrogen, and stored at -85°C. Total RNA was extracted by using RNAzol (Tel-Test, TX). The amount of RNA was determined from the absorbance at 260 nm using a Gilford Response Spectrophotometer. The oligomer probes used to assess P450 IAl, lA2, IIEl, IIBl and IIB2 mRNAs have the sequences of 5'-TCTGGTGAGCATCCAGGACA-3',5'-GGAAAAGGAACAAGGGTGGC-3', 5'-TCCAGTGACTGAAGGTGTTCCTT-3' 5'-GGTTGGTAGCCGGTGTGA-3' and 5'GGATGGTGGCCTGTGAGA-3', respectively (21, 22,231. 18s ribosomal RNA probe has a sequence of S-CACCTCTAGCGGCGCAATAC-Y (24). The probes were custom-synthesized by Genosys Biotechnologies, Inc. (Woodlands, TX) and were 5’-end labeled with (Y32~) ATP (specific activity, 6000 Ci/m mol; New England Nuclear, MA) and T4polynucleotide kinase (New England Biolabs, MA). The specific activities of the oligomers used for hybridization were typically 2 9x106 cpm/pmol. For northern blot analysis, 20 pg of total RNA were denatured (65”C/lO min/2.2M formaldehyde), subjected to electrophoresis at 46V on 1.2 % agarose/2.2M formaldehyde gels, transferred to nylon filters (Zeta-probe, Bio-Rad, CA) in HETS (Tel-Test) and immobilized by UV irradiation (Stratagene, UV Stratalinker 2400). Prehybridization and hybridization were performed according to Bio-Rad Zeta-Probe Blotting Membranes Instruction Manual except that pH of the perhybridizing solution was adjusted to 7.0 and 10% dextran sulfate was included in both of these solutions. Autoradiography was carried out after exposure to Kodak X-OMAT AR (XAR) film at -85°C for 48 hrs. Densitometric measurement was performed by using Bio-Rad Model 620 Densitometer. Recombinant human IL-1 a (lot 117-271) was generously provided by Drs. Alvin Stern, Swapan Roy and Peter Lomedico of Hoffmann-La Roche, Nutley, NJ. This lot of IL-1 had a specific activity of 3 x 108 units/mg protein as measured by the DlO assay and contained small amounts of endotoxin (0.74 EU/mg protein). NADPH, NADH, cytochrome c, superoxide dismutase, 7-hydroxycoumarin and benzphetamine were purchased from Sigma Chemical (St. Louis, MO). 7ethoxycoumarin was purchased from Pierce (Rockford, IL). All other chemicals were of analytical grade. Statistical analysis was performed on a Macintosh II with the use of Stat View software. Statistical significance of the difference between concurrent control and treated groups was determined by one-way analysis of variance and the Dunnet t test. RESULTS AND DISCUSSION After IL-1 administration P450 was decreased to 54% (34.8/64.7) of the control value at 24 hours and recovered to 64% (36.6/57.4) at 72 hours when results were expressed on per mg protein basis. Cytochrome bg showed significant decrease to 73% (68.5/44.0) at 24 hours and recovered to 92% (69.7/76.1) at 72 hours. The activity 1085
Vol.
185,
No.
3,
Table
1992
1. Effect
BIOCHEMICAL
of recombinant
AND
Inledeukln-1
BIOPHYSICAL
treatment
RESEARCH
on lung mlcrosomal
COMMUNICATIONS
enzymes
roteln) -YF=-%iT l/m
) 5 (/lun
(3)
60.3 f18.7
1.17 f0.33
82.9 f14.9
1.62 f0.34
87.6 f6.0
1.71 fO.08
3.06 f0.08
59.6 f2.0
Saline
2411t (3)
64.7 f 8.6
1.38 io. 19
94.0 f 4.3
2.00 10.10
81.0 rt5.0
1.72 f0.04
2.86 f0.13
60.7 f1.3
Interleutdn-1
24ht 13)
34.89 f 7.0
0.72’ tcO.23
68.5’ + 4.4
1.38’ fO.10
84.7 f7.5
1.70 1tO.08
2.91 to.24
58.3 f3.3
Saline
72ht (3)
57.4 f20.5
1.21 50.44
76.1 * 7.7
1.61 f0.27
84.6 f2.2
1.78 f0.08
2.89 fO.ll
60.7 f2.1
Interleuktn-1
72ht (41
36.6 f 9.0
0.84 f0.22
69.7 ill.2
1.62 f0.42
79.6 rt7.3
1.83 f0.21
2.73 f0.20
62.7 f8.7
Treament h) Untreated
control
rotein]
[/lung)
Values are the meatiSD. t Hours after recombinant Interleuktn 1 or saline InJectIon. 8 Cytochrome P450 and cytochrome bs content are expressed as pmol 19 NADPH-cytochrome P45O(c) reductase acttvlty Is expressed as nmol mtcrosomat proletn and pmol of cytochrome c reduced per mm per ~$1 NADH-cytochrome bs(fenicyanlde) reductase actlvlty Is expressed of mlcrosomal protetn or lung. Signlftcantly different from concurrent control group, l P< 0.05
(/mg
I’450 F&.&J&& protein) [/lung)
(/mg
bc, P&.&m proteln) (/lung)
per mg of mlcrosomal protein and nmol per lung. of cytochrome c reduced per mln per mg of lung. as pmol of ferrlcyantde reduced per mm per mg
of NADPH P450 reductase or NADH b5 reductase were unaltered (Table 1). This depression of I’450 in the lungs was more pronounced than the degree of depression seen in the liver after the identical treatment with IL-1 (Table 3). This difference is probably due to selectivity of the action of IL-1 towards different I’450 isozymes and to the difference of the constituitive P450 isozymes between the lung and the liver. In the liver, the major constituitive enzyme is P450 IICll which are unaffected by the action of IL-1 (25). In the lung the major forms are I’450 IVB and P450 IIBl (26) and I?450 IA1 has only been found in trace amounts. The degree of depression of pulmonary I’450 content of up to 50% indicates that both I’450 IIBl and I’450 IVB are likely to be reduced by the action of IL-l. The activity of 7-ECOD and BPND was reduced to about 75% and 60% of control value at both 24 and 72 hours, respectively (data not shown). The activity of 7-ECOD is largely attributed to I’450 IA1 with minor contributions from I’450 IIBl and I’450 IVB (26, 27). Our data suggests that reduced amounts of any of three I’450 isozymes may produce this degree of depression. BPND activity is largely attributed to I’450 IIBl (27) and the decrease in this catalytic activity suggests that I?450 IIBl is decreased although no information is available on the role of I’450 IVB in BPND activity. Figure 1 shows Northern blot analysis of I’450 IIBl mRNA along with the results of 18s ribosomal RNA hybridization. We have used mRNA probes for P450 IAl, IA2, IIB2, IIEl in addition to I?450 IIBl probe. However, we were unsuccessful in obtaining signals for mRNA except I?450 IIBl. I’450 IAl, however, has been demonstrated in the lungs of rats with polymerase chain reaction amplification (24). All these probes, however, were successfully utilized for hepatocytes (25). As 1086
Vol.
185,
No.
3,
BIOCHEMICAL
1992
AND
BIOPHYSICAL
12
control
RESEARCH
COMMUNICATIONS
48 0-4
2.0 Kb-
P450
IlBl
18s
rRNA
Figure 1. Northern blot analysis of rat pulmonary P450 IIBl after treatment with IL-l. Twenty lg of total pulmonary RNA per lane from control and experimental group
were
subjected to northern
migration
of P450 IIBl
blot analysis. The arrow indicates the position of
mRNA.
18s rRNA
oligonucleotide
probe
is used as loading
control. shown,
there was
considerable
after administration density
indicated
of IL-l.
depression
of I’450 IIBl mRNA
Densitometric
that I’450 IIBl
mRNA
analysis
after correction
was reduced
values 12 and 48 hours after IL-l administration Figure 2 shows the changes of protein
at 24 and 72 hours for 18s RNA
to 77% and 69% of control
respectively (data not shown). content of P450 IIBl. Densitometric
measurement indicate that there was 20% decrease in protein content at both 24 and 72 hours after IL-l administration (data not shown). These data indicate that IL-l administration content
results
by 24 hours.
monoxygenase
I’450 IIBl
mRNA
These results are consistent
by 12 hours
with
the results
from
microsomes
and protein of preceeding
study.
Superoxide hours after IL-l
in decreased
anion generation administration
was measured
and was expressed per microsomal
at 24 and 72
protein
and per
lung (Table 2). At 24 hours superoxide anion was reduced to 73% (2&O/38.2) of control value (~~0.01) and at 72 hours recovered to 90% (32.8/36.3) of control (p.CO.05). Since liver is the major source of P450, the alterations superoxide
generation
was also studied. Control
on hepatic
I’450 and
Table 3 shows that hepatic cytochrome
24
P450
72 (W
---
cP450
II61
Figure 2. Western blot analysis of pulmonary microsomes from control and IL-l treated rats. Microsomes (2 l.tg protein in each lane) were subjected to SDS polyacrylamide gel (10% gel) electrophoresis followed by electrophoretic transfer to nitrocellulose membranes. The membranes were incubated with monoclonal antibody against P450 IIBl and stained using alkaline phosphatase conjugated second antibody. Each lane represents one rat. 1087
Vol.
BIOCHEMICAL
185, No. 3, 1992
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
Table 2. Effect of recombinant interleukin-1 treatment on lung microsomal superoxide generating activity Treament (n) Untreated Saline Interleukin Saline
Superoxide generating activW$ (/mg protein) (/lung)
control (31
1.78 kO.07
34.6 k1.7
24ht 131
1.79 to.14
38.2 k2.8
1.39** 20.03
28.0** 53.3
1.72 kO.06
36.3 k3.5
1 24ht (31 72ht (3)
Interleukin
1 72ht 1.44* 32.8 kO.22 rt3.3 (41 Values are the me&SD. t Hours after recombinant interleukin 1 or saline injection. 5 Activity is expressed as nmol per min per mg microsomal protein or lung. Significantly different from concurrent control group, *PC 0.05. **P
