Vol.
187,
No.
September
3, 1992
30,
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
1992
COMMUNICATIONS Pages
1262-l
269
DIFFERENTIAL OXIDASE ACTIVITY OF HEPATIC AND PULMONARY MICROSOMAL CYTOCHROME P-450 ISOZYMES AFTER TREATMENT WITH CYTOCHROME P-450 INDUCERS Hironori IDepartment 2Laboratory Received
Sakai,l Sang S. Park2 and Yutaka Kikkawal
of Pathology, of Comparative
August
14,
College of Medicine, University Irvine, California 92717 Carcinogenesis,
NCI-FCRDC,
of California,
Frederick,
MD
Irvine 21702-1201
1992
SUMMARY
Phenobarbital, 3-methylcholanthrene, acetone and pyrazole were used of cytochrome P450 and the NADPH-dependent oxidase activity (O-2 production) of pulmonary and hepatic microsomes was determined. Oxidase activity of microsomes from 3-methylcholanthrene-treated rats was significantly decreased as compared to that of controls when expressed on the basis of cytochrome P450 content (30% decrease for liver, 60% decrease for lung). The oxidase activity of liver microsomes from pyrazole-treated rats showed a significant increase, whereas phenobarbital treated microsomes had average superoxidegenerating activity. The contribution of cytochromes CYP lA, CYP 2B and CYP 2El to superoxide-generating activity was investigated using monoclonal antibodies. Monoclonal antibody 1-91-3 against CYP 2El inhibited superoxide generation by 58% in liver microsomes from pyrazole-treated rats. Monoclonal antibodies l-7-1 and 266-3 against CYP 1A and CYP2B, respectively, had no effect on superoxide generation. These results indicate that different cytochrome I’450 isoforms are mainly responsible for differential superoxide generating activities of microsomes and complement the reconstitution study of Morehouse and Aust. Furthermore, our study indicates that CYP lA1, induced by 3-MC, demonstrates an unusually low oxidase activity. 0 1992Academic Press,Inc. as inducers
The cytochrome reductase, responsible substrates.
P450 system consists of P45Os, cytochrome
b5, NADPH
I’450
NADH cytochrome b5 reductase, membrane phospholipids and is for the metabolism of various drugs, xenobiotics and endogenous During its oxidation of substrates, this electron transport system has been
demonstrated to produce Reduced concentration
reactive oxygen species (ROS) such as 02- and Hz02 (l-3). of I’450 (4, 5) induced by interferon-inducers (4) or
interleukin-1, (5) has been shown to be associated with prolonged survival of rats in otherwise lethal hyperoxia. Rats treated with these cytokines generate lesser amounts of superoxide anion as compared to the untreated controls (7-8). Mice genetically refractory to induction of cytochrome I’450 by aromatic hydrocarbons or by hyperoxia 0006-291X/92
Copyright All rights
live longer after exposure to >95% 02, as compared $4.00
0 I992 by Academic Press, of reproduction in any form
Inc. reserved.
1262
to the ones which
Vol.
187,
induce
No.
3,
1992
BIOCHEMICAL
AND
I?450 by these hydrocarbons
acetone prior
to hyperoxic
BIOPHYSICAL
or by hyproxia
exposure
RESEARCH
(9).
COMMUNICATIONS
Induction
of CYP 2El by
results in a decrease of survival
time in adult
rats (10). These observations indicate that reduced I’450 and attendant reduction of superoxide anion from which various ROS derive, are beneficial under hyperoxia. On the other hand, studies by Mansour et al. (11) showed that adult rats survive
longer
upon
hyperoxic
exposure
after
treatment
with
well-known
cytochrome I’450 inducers such as 3-MC and P-NF. This study appears to contradict other observations cited above. Because of these two apparently
contradictory
observations,
we hypothesized
that different cytochrome P450 inducers induce different cytochrome I’450 forms with varying oxidase activities, and that 3-MC or B-NF might induce I’450 isozymes of low oxidase activity. We examined this by determining the NADPH-dependent oxidase activity of pulmonary and hepatic microsomes thus treated. The results presented here demonstrate, for the first time, that CYP 1Al which 3-MC, markedly lower the level of oxidase activity. MATERIALS
AND
is inducible
with
METHODS
Adult Sprague Dawley CD strain, viral-antigen and pathogen-free male rats (body weight, 300-350g) were obtained from Harlan-Sprague Dawley (Indianapolis, IN). Various I’450 isoforms were induced as follows: 3-MC (Sigma) dissolved in corn oil and sodium phenobarbital (Sigma) in 0.9% NaCl were injected i.p. daily for 4 days at 25 mg and 80 mg per kg of body weight, respectively. Pyrazole (Sigma) in 0.9% NaCl was also given i.p. daily for 4 days at a dose of 200 mg per kg of body weight. Acetone (5%, v/v) was given for 10 days in place of drinking water. During the course of these experiments, rats were allowed rodent laboratory chow and water ad libitum and kept on a 12 hour light on/off cycle. Control animals consisted of Animals were injection with appropriate carriers and those with no treatment. sacrificed, and microsomes from livers and lungs were obtained as described previously (8), and stored at -85°C. Protein, P450 and b5 contents, I’450 reductase and b5 reductase activity were determined as described earlier (8). The 0-deethylation of 7-ethoxycoumarin (7ECOD) and benzphetamine N-demethylase activity (BPND) and aniline hydroxylation (AH) were measured as described previously (7, 8). Acetylated cytochrome c was used for the measurement of superoxide (02-) production (12). The difference in the rates of acetylated cytochrome c reduction measured in the presence or absence of SOD (Sigma) was used as a measure of the amount of 02- generated. Three monoclonal antibodies were used to characterize the superoxidegenerating activity in microsomes. These monoclonal antibodies have been shown to be specific against cytochrome P450 isozymes as follows: MAb 2-66-3 to CYP 2Bl and 2B2, MAb 1-7-1 to CYP 1Al and lA2, and MAb 1-91-3 to CYP 2El. As a control, MAb HyHel-9 against chicken lysosome was used. MAbs were incubated with microsomes (1OOpg of protein) and buffer at room temperture for 30 min before starting the reaction by the addition of acetylated cytochrome c and NADPH. In preliminary experiments, maximal inhibition by MAbs were obtained at a MAb protein/microsomal protein ratio of about 3.0 for assays of drug metabolism. Therefore, 35Opg of MAb were used in these assays. 1263
Vol.
187,
No.
3,
BIOCHEMICAL
1992
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
Statistical analysis was performed on a Macintosh II using Stat-View 512+ software. Statistical significances of the differences between untreated control and treated groups were determined by a one-way analysis of variance and the Dunnett t-test. There were no significant differences between untreated control and saline or oil control groups. Therefore, only statistical significances between untreated control and I’450 inducer-treated groups are presented. RESULT AND Table reductase
1 shows
that
after
DISCUSSION
phenobarbital
treatment,
both
P450 and
I’450
increased
10 and 60%, respectively, while b5 and b5 reductase were not With 3-MC treatment, I’450 and bg significantly increased, significantly altered. while I’450 reductase and b5 reductase tended to decrease. With administration of acetone, all four components showed significant increase whereas with pyrazole all of these showed
significant
Phenobarbital
decrease.
(PB) treatment
increased the activities of BPND and 7-ECOD by
100% and 450%, respectively (Table 2). PB induces hepatic CYP 2Bl and 2B2, while CYl? 2Cll is reduced (13). BPND activity represents CYP 2Bl and 2Cll at 100 and 40 on an equimolar proportion
basis (13).
Since uninduced
hepatic microsomes
of CYP 2B (-1%) and a large proportion
of CYP 2Cll
contain
a small
(43%), BPND activity
of control microsomes should represent the activity generated by CYP 2Cll. With PB an increase of CYP 2Bl to 48% and a decrease of CYP 2Cll (14) probably accounts for the modest increase of BPND activity in hepatic microsomes seen in this study. 7-ECOD activity is catalyzed by CYP 1Al and 2Bl at the ratio of 100:34 on an equimolar
Table
1.
basis (13). Thus, in the absence of any significant
Microsomal
Enzymes N
of the livers Cytochrome
from P450t
rats
treated
with
Cylochrome
various bst
quantities
cytochrome
of CYP 1Al
P450
inducers
NADPH-P450tt reductase
NADH-bSttt reductase
Untreat-Control
7
1.373tO.088
0.584f0.047
0.189f0.026
5.268kO.453
Phenobarbital
7
2.706iO.190”
0.679f0.068
0.3QO+O.O25”
4.788f0.376
3-MC
7
2.141f0.162”
0.759f0.059”
0.163+0.016
4.547f0.357’
Acetone
7
1.66810.186”
0.908f0.113”
0.263f0.038”
6.62710.576”
Pyrazole
6
1.071f0.194’
0.333f0.043”
0.146kO.018’
3.621f0.405”
t Cytochrome P450 and cytochrome bs content are expressed as nmol per mg of microsomal protein. tt NADPH-cytochrome P45O(c) reductase activity is expressed as .ttmol of cytochrome c reduced per mIn per mg of mkrosomal protein. ttt NADH-cytochrome b5(ferricyanide) reductase activity is expressed as pmol of ferricyanide reduced per min per mg of microsomal protein. Values are the meant SD. l PlAl. Our results are consistent with additional information that microsomes obtained lesser quantitites microsomes.
with
CYP lA1,
anion
MAb 1-91-3 (anti CYP 2El) inhibited in liver microsomes from pyrazole-treated
than
anion production: CYP this earlier observation and add from 3-MC-treated rats produce
those
produced
by
untreated
superoxide production by 42% (2.62/4.54) rats (Table 3). Demonstration that MAb
1-91-3 decreased AH activity by 60% (data not shown) and superoxide anion generation by 40% suggests that after pyrazole treatment, CYP 2El may constitute at least 30-35% of total P450 content of hepatic microsomes, after accounting for higher oxidase activity of CY’P 2El. MAb 1-7-1 (anti CYI? 1A) and MAb 2-66-3 (anti CYI? 2B) did not inhibit superoxide generation in all three treated microsomes. MAb 2-66-3 enhanced superoxide-generating activity by 54% in microsomes from 3-MC-treated rats. We do not know the reason for this enhancement of superoxide generation. Enhancement of drug metabolism, however, has been demonstrated with this antibody (18). Monoclonal antibodies we used in this experiment are an effective 1266
Vol.
187,
Table
No.
3,
3. Inhibition
1992
BIOCHEMICAL
of superoxide
generating
AND
activity
Phenobarbital
9
2-66-3
10.54+1.65(100)
3-Methylcholanthrene Pyrazole
RESEARCH
by monoclonal
Monoclonal My-He1
BIOPHYSICAL
Antibodies
antibodies
9.25+2.62(88)
1-7-1
liver
microsomes
of P450)
(P45OlA)
1-91-3
(P450IIEl)
11.67+1.56(111)
9.19?1.40(87)
5.11+0.42(100)
7.88+0.81(154)*
5.80+0.81(114)
6.03+1.40(118)
4.54+1.00(100)
4.74kO.99(104)
4.23+1.04(93)
2.62*0.80(58)’
Values represent remaining superoxide generating activity ( nmol/ and, in parentheses, the % remaining activity expressed as (activity Values are the mean + SD. N=4 for each group. “P
187,
No.
September
3, 1992
30,
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
1992
COMMUNICATIONS Pages
1262-l
269
DIFFERENTIAL OXIDASE ACTIVITY OF HEPATIC AND PULMONARY MICROSOMAL CYTOCHROME P-450 ISOZYMES AFTER TREATMENT WITH CYTOCHROME P-450 INDUCERS Hironori IDepartment 2Laboratory Received
Sakai,l Sang S. Park2 and Yutaka Kikkawal
of Pathology, of Comparative
August
14,
College of Medicine, University Irvine, California 92717 Carcinogenesis,
NCI-FCRDC,
of California,
Frederick,
MD
Irvine 21702-1201
1992
SUMMARY
Phenobarbital, 3-methylcholanthrene, acetone and pyrazole were used of cytochrome P450 and the NADPH-dependent oxidase activity (O-2 production) of pulmonary and hepatic microsomes was determined. Oxidase activity of microsomes from 3-methylcholanthrene-treated rats was significantly decreased as compared to that of controls when expressed on the basis of cytochrome P450 content (30% decrease for liver, 60% decrease for lung). The oxidase activity of liver microsomes from pyrazole-treated rats showed a significant increase, whereas phenobarbital treated microsomes had average superoxidegenerating activity. The contribution of cytochromes CYP lA, CYP 2B and CYP 2El to superoxide-generating activity was investigated using monoclonal antibodies. Monoclonal antibody 1-91-3 against CYP 2El inhibited superoxide generation by 58% in liver microsomes from pyrazole-treated rats. Monoclonal antibodies l-7-1 and 266-3 against CYP 1A and CYP2B, respectively, had no effect on superoxide generation. These results indicate that different cytochrome I’450 isoforms are mainly responsible for differential superoxide generating activities of microsomes and complement the reconstitution study of Morehouse and Aust. Furthermore, our study indicates that CYP lA1, induced by 3-MC, demonstrates an unusually low oxidase activity. 0 1992Academic Press,Inc. as inducers
The cytochrome reductase, responsible substrates.
P450 system consists of P45Os, cytochrome
b5, NADPH
I’450
NADH cytochrome b5 reductase, membrane phospholipids and is for the metabolism of various drugs, xenobiotics and endogenous During its oxidation of substrates, this electron transport system has been
demonstrated to produce Reduced concentration
reactive oxygen species (ROS) such as 02- and Hz02 (l-3). of I’450 (4, 5) induced by interferon-inducers (4) or
interleukin-1, (5) has been shown to be associated with prolonged survival of rats in otherwise lethal hyperoxia. Rats treated with these cytokines generate lesser amounts of superoxide anion as compared to the untreated controls (7-8). Mice genetically refractory to induction of cytochrome I’450 by aromatic hydrocarbons or by hyperoxia 0006-291X/92
Copyright All rights
live longer after exposure to >95% 02, as compared $4.00
0 I992 by Academic Press, of reproduction in any form
Inc. reserved.
1262
to the ones which
Vol.
187,
induce
No.
3,
1992
BIOCHEMICAL
AND
I?450 by these hydrocarbons
acetone prior
to hyperoxic
BIOPHYSICAL
or by hyproxia
exposure
RESEARCH
(9).
COMMUNICATIONS
Induction
of CYP 2El by
results in a decrease of survival
time in adult
rats (10). These observations indicate that reduced I’450 and attendant reduction of superoxide anion from which various ROS derive, are beneficial under hyperoxia. On the other hand, studies by Mansour et al. (11) showed that adult rats survive
longer
upon
hyperoxic
exposure
after
treatment
with
well-known
cytochrome I’450 inducers such as 3-MC and P-NF. This study appears to contradict other observations cited above. Because of these two apparently
contradictory
observations,
we hypothesized
that different cytochrome P450 inducers induce different cytochrome I’450 forms with varying oxidase activities, and that 3-MC or B-NF might induce I’450 isozymes of low oxidase activity. We examined this by determining the NADPH-dependent oxidase activity of pulmonary and hepatic microsomes thus treated. The results presented here demonstrate, for the first time, that CYP 1Al which 3-MC, markedly lower the level of oxidase activity. MATERIALS
AND
is inducible
with
METHODS
Adult Sprague Dawley CD strain, viral-antigen and pathogen-free male rats (body weight, 300-350g) were obtained from Harlan-Sprague Dawley (Indianapolis, IN). Various I’450 isoforms were induced as follows: 3-MC (Sigma) dissolved in corn oil and sodium phenobarbital (Sigma) in 0.9% NaCl were injected i.p. daily for 4 days at 25 mg and 80 mg per kg of body weight, respectively. Pyrazole (Sigma) in 0.9% NaCl was also given i.p. daily for 4 days at a dose of 200 mg per kg of body weight. Acetone (5%, v/v) was given for 10 days in place of drinking water. During the course of these experiments, rats were allowed rodent laboratory chow and water ad libitum and kept on a 12 hour light on/off cycle. Control animals consisted of Animals were injection with appropriate carriers and those with no treatment. sacrificed, and microsomes from livers and lungs were obtained as described previously (8), and stored at -85°C. Protein, P450 and b5 contents, I’450 reductase and b5 reductase activity were determined as described earlier (8). The 0-deethylation of 7-ethoxycoumarin (7ECOD) and benzphetamine N-demethylase activity (BPND) and aniline hydroxylation (AH) were measured as described previously (7, 8). Acetylated cytochrome c was used for the measurement of superoxide (02-) production (12). The difference in the rates of acetylated cytochrome c reduction measured in the presence or absence of SOD (Sigma) was used as a measure of the amount of 02- generated. Three monoclonal antibodies were used to characterize the superoxidegenerating activity in microsomes. These monoclonal antibodies have been shown to be specific against cytochrome P450 isozymes as follows: MAb 2-66-3 to CYP 2Bl and 2B2, MAb 1-7-1 to CYP 1Al and lA2, and MAb 1-91-3 to CYP 2El. As a control, MAb HyHel-9 against chicken lysosome was used. MAbs were incubated with microsomes (1OOpg of protein) and buffer at room temperture for 30 min before starting the reaction by the addition of acetylated cytochrome c and NADPH. In preliminary experiments, maximal inhibition by MAbs were obtained at a MAb protein/microsomal protein ratio of about 3.0 for assays of drug metabolism. Therefore, 35Opg of MAb were used in these assays. 1263
Vol.
187,
No.
3,
BIOCHEMICAL
1992
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
Statistical analysis was performed on a Macintosh II using Stat-View 512+ software. Statistical significances of the differences between untreated control and treated groups were determined by a one-way analysis of variance and the Dunnett t-test. There were no significant differences between untreated control and saline or oil control groups. Therefore, only statistical significances between untreated control and I’450 inducer-treated groups are presented. RESULT AND Table reductase
1 shows
that
after
DISCUSSION
phenobarbital
treatment,
both
P450 and
I’450
increased
10 and 60%, respectively, while b5 and b5 reductase were not With 3-MC treatment, I’450 and bg significantly increased, significantly altered. while I’450 reductase and b5 reductase tended to decrease. With administration of acetone, all four components showed significant increase whereas with pyrazole all of these showed
significant
Phenobarbital
decrease.
(PB) treatment
increased the activities of BPND and 7-ECOD by
100% and 450%, respectively (Table 2). PB induces hepatic CYP 2Bl and 2B2, while CYl? 2Cll is reduced (13). BPND activity represents CYP 2Bl and 2Cll at 100 and 40 on an equimolar proportion
basis (13).
Since uninduced
hepatic microsomes
of CYP 2B (-1%) and a large proportion
of CYP 2Cll
contain
a small
(43%), BPND activity
of control microsomes should represent the activity generated by CYP 2Cll. With PB an increase of CYP 2Bl to 48% and a decrease of CYP 2Cll (14) probably accounts for the modest increase of BPND activity in hepatic microsomes seen in this study. 7-ECOD activity is catalyzed by CYP 1Al and 2Bl at the ratio of 100:34 on an equimolar
Table
1.
basis (13). Thus, in the absence of any significant
Microsomal
Enzymes N
of the livers Cytochrome
from P450t
rats
treated
with
Cylochrome
various bst
quantities
cytochrome
of CYP 1Al
P450
inducers
NADPH-P450tt reductase
NADH-bSttt reductase
Untreat-Control
7
1.373tO.088
0.584f0.047
0.189f0.026
5.268kO.453
Phenobarbital
7
2.706iO.190”
0.679f0.068
0.3QO+O.O25”
4.788f0.376
3-MC
7
2.141f0.162”
0.759f0.059”
0.163+0.016
4.547f0.357’
Acetone
7
1.66810.186”
0.908f0.113”
0.263f0.038”
6.62710.576”
Pyrazole
6
1.071f0.194’
0.333f0.043”
0.146kO.018’
3.621f0.405”
t Cytochrome P450 and cytochrome bs content are expressed as nmol per mg of microsomal protein. tt NADPH-cytochrome P45O(c) reductase activity is expressed as .ttmol of cytochrome c reduced per mIn per mg of mkrosomal protein. ttt NADH-cytochrome b5(ferricyanide) reductase activity is expressed as pmol of ferricyanide reduced per min per mg of microsomal protein. Values are the meant SD. l PlAl. Our results are consistent with additional information that microsomes obtained lesser quantitites microsomes.
with
CYP lA1,
anion
MAb 1-91-3 (anti CYP 2El) inhibited in liver microsomes from pyrazole-treated
than
anion production: CYP this earlier observation and add from 3-MC-treated rats produce
those
produced
by
untreated
superoxide production by 42% (2.62/4.54) rats (Table 3). Demonstration that MAb
1-91-3 decreased AH activity by 60% (data not shown) and superoxide anion generation by 40% suggests that after pyrazole treatment, CYP 2El may constitute at least 30-35% of total P450 content of hepatic microsomes, after accounting for higher oxidase activity of CY’P 2El. MAb 1-7-1 (anti CYI? 1A) and MAb 2-66-3 (anti CYI? 2B) did not inhibit superoxide generation in all three treated microsomes. MAb 2-66-3 enhanced superoxide-generating activity by 54% in microsomes from 3-MC-treated rats. We do not know the reason for this enhancement of superoxide generation. Enhancement of drug metabolism, however, has been demonstrated with this antibody (18). Monoclonal antibodies we used in this experiment are an effective 1266
Vol.
187,
Table
No.
3,
3. Inhibition
1992
BIOCHEMICAL
of superoxide
generating
AND
activity
Phenobarbital
9
2-66-3
10.54+1.65(100)
3-Methylcholanthrene Pyrazole
RESEARCH
by monoclonal
Monoclonal My-He1
BIOPHYSICAL
Antibodies
antibodies
9.25+2.62(88)
1-7-1
liver
microsomes
of P450)
(P45OlA)
1-91-3
(P450IIEl)
11.67+1.56(111)
9.19?1.40(87)
5.11+0.42(100)
7.88+0.81(154)*
5.80+0.81(114)
6.03+1.40(118)
4.54+1.00(100)
4.74kO.99(104)
4.23+1.04(93)
2.62*0.80(58)’
Values represent remaining superoxide generating activity ( nmol/ and, in parentheses, the % remaining activity expressed as (activity Values are the mean + SD. N=4 for each group. “P
