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Vol. 28, Issue 9, 1112-1120, September 2000
Département de Pharmacologie, Faculté de Médecine, Université de Montréal Montréal, Québec, Canada
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Abstract |
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 Top
 Abstract
 Introduction
Materials and Methods
Results
Discussion
References
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Serum of rabbits with a turpentine-induced acute inflammatory reaction (RSINFLA) and serum of humans with a viral infection (HSINF) were previously shown to diminish hepatic cytochrome P450 (P450) content and activity. To document the role of reactive oxygen intermediates in the serum-mediated decrease in P450 content and activity, hepatocytes of rabbits with an acute inflammatory reaction (HINFLA) were incubated with RSINFLA and HSINF for 4 h, and total P450 content (spectrally measurable P450), P450 activity (assessed by estimating the formation of theophylline metabolites), and amount of CYP1A1, CYP1A2, and CYP3A6 proteins were measured. RSINFLA or HSINF decreased P450 content and activity without affecting the amount of CYP1A1 and -1A2 HINFLA. Exposure of HCONT or HINFLA to hydrogen peroxide (0.01-1.0 mM) and sodium nitroprusside (0.01-1.0 mM) produced a dose-dependent decrease in P450 content and in the formation of theophylline metabolites without modifying the amount of CYP1A1 and CYP1A2, whereas lipid peroxidation increased. Incubation of L-NAME (0.05-1.0 mM), dimethylthiourea (6.25-50 mM), or N-acetylcysteine (0.01-1.0 mM) with HINFLA partially prevented the decrease in P450 content and activity and the increased lipid peroxidation induced by RSINFLA and HSINF. On the other hand, 3-amino-1,2,4-triazole (10-100 mM) or diethyldithiocarbamate (1.0-10 mM) potentiated RSINFLA- and HSINF-mediated decreases in P450 content and activity and the increase in lipid peroxidation, without affecting the amount of CYP1A1 or -1A2; DL-buthionine-(S,R)-sulfoximine (2.5-25 mM) potentiated only the inhibition of 1,3-dimethyluric acid formation. It is concluded that reactive oxygen intermediates are implicated in the decrease of HINFLA P450 content and activity induced by 4 h of exposure to RSINFLA or HSINF.
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Introduction |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Endotoxins, sepsis, and acute inflammatory
reactions down-regulate hepatic cytochrome P450
(P450)1 apoproteins
through transcriptional and post-transcriptional mechanisms (Morgan,
1989
). However, the cascade of events leading to the down-regulation of
P450 remains poorly defined. Reactive oxygen intermediates (ROI),
induced by the inflammatory reaction, have been
implicated in the signaling pathway leading to P450 down-regulation in
vivo (Galal and du Souich, 1999) and in vitro (Chen et al., 1995).
Hydrogen peroxide (H2O2) is
able to diminish the expression of selected isozymes of hepatic P450
(Barker et al., 1994), and
H2O2 mediates the
depression of P450 by interferon-
(IFN-) (Moochhala and Renton,
1991). Moreover, nitric oxide synthase (NOS) inhibitors prevent the
decrease in P450 activity mediated by cytokines or lipopolysaccharides
in vivo (Khatsenko et al., 1993) and in vitro (Carlson and Billings,
1996). Furthermore, the addition of nitric oxide (NO·)
generators to microsomes (Khatsenko et al., 1993) or cultured hepatocytes (Carlson and Billings, 1996) down-regulates hepatic P450.
Potentially conflicting with these reports, it has been shown that
xanthine oxidase is not involved in the P450 down-regulation induced by
IFN- or poly(IC) (Cribb and Renton, 1993). On the other hand, NOS
inhibitors do not prevent cytokine-induced down-regulation of CYP2C11
protein and mRNA (Sewer and Morgan, 1997) or the reduction in P450
content (Hodgson and Renton, 1995). In addition, in vivo the
administration of NO· generators does not appear to
down-regulate P450 (Hodgson and Renton, 1995).
Forty-eight hours after the production of an inflammatory reaction by
the s.c. injection of turpentine, hepatic CYP1A1, -1A2, and -3A6 are
down-regulated (Kurdi et al., 1999). The serum from these rabbits with
a turpentine-induced inflammatory reaction (RSINFLA) and the serum from humans with an upper
respiratory viral infection (HSINF) when
incubated for 4 h with hepatocytes from rabbits with a
turpentine-induced inflammatory reaction (HINFLA) reduce P450 content and activity. The RSINFLA-
and HSINF-induced decrease in P450 content is
directly associated with an increase in lipid peroxidation, suggesting
that ROI are implicated in the decrease in P450 content (El-Kadi et
al., 1997).
The present study aimed to investigate whether ROI are implicated in
the RSINFLA- and
HSINF-mediated decrease in hepatic P450 content
and activity. For this purpose, we have 1) determined the effect of
H2O2 and NO· on the
amount and activity of P450, 2) assessed whether the addition of
L-NAME and antioxidants to hepatocytes prevent the decrease in P450 content and activity mediated by RSINFLA
and HSINF, and 3) examined whether the addition
of inhibitors of antioxidant enzymes to hepatocytes potentiate the
decrease in P450 content and activity mediated by
RSINFLA and HSINF. P450
activity was assessed by measuring the metabolism of theophylline. In
rabbits, theophylline is transformed to 1,3-dimethyluric acid
(1,3-DMU), 3-methylxanthine (3-MX), and 1-methyluric acid (1-MU)
essentially by CYP1A1, -1A2, and more marginally by CYP3A6 (Kurdi et
al., 1999).
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Materials and Methods |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Experimental Protocol.
Male New Zealand rabbits (1.8-2.2 kg) were obtained from the Ferme
Cunicole (St. Valérien, Québec). Rabbits were housed in
separate cages and fed water and chow ad libitum for at least 7 days
before being used. The inflammatory reaction was induced locally by
injecting turpentine (5 ml) s.c. at two distinct sites on the back of
the rabbits (Parent et al., 1992). Control rabbits received 5 ml of
sterile NaCl 0.9% s.c. at two sites of the back. The severity of the
inflammatory reaction was assessed by measuring the concentration of
seromucoids. Seromucoids are acute phase proteins, mostly
1-glycoprotein acid, and in minor amounts
1-antitrypsin and haptoglobulin. They were
assayed by means of a colorimetric reaction with copper and folin
phenol reagents yielding a maximal absorbance at 540 nm (Parent et al.,
1992). All the experiments were conducted according to the Canadian
Council on Animal Care guidelines for use of laboratory animals.
, with minor modifications (El-Kadi et al., 1997).
Hepatocytes (4 × 106/ml) were placed into
12-well plastic culture plates (Falcon, Becton Dickinson Labware,
Lincoln Park, NJ) coated with Type I rat-tail collagen (Sigma Chemical
Company, St. Louis, MO); cells were suspended in William's medium E
(Sigma) supplemented with 10% calf serum and insulin 1 µM
(Boehringer Mannheim GmbH, Germany). The plastic culture plates were
incubated at 37°C in a humidifier with 95% O2
and 5% CO2. Viability was assessed before and
after the incubation period by the trypan blue (0.2%) exclusion
method, and in both instances the viability was over 90%.
In all experiments, the effect of the experimental conditions on
hepatic P450 was characterized by culturing hepatocytes with serum for
4 h, and assessing the catalytic activity of the P450 using
theophylline as a probe. The experiments were initiated by adding 100 µl of serum and 50 µl of theophylline (Sigma), dissolved in
serum-free William's medium E, to 1 ml of incubation media containing
freshly isolated 4 × 106 hepatocytes per
well. The final concentration of theophylline was 176 µM. At time
zero, 350 µl of the supernatant was collected from each well (control
sample), and following 4 h of incubation, the remaining medium was
collected and frozen at 
20°C until the theophylline metabolites
3-MX, 1-MU, and 1,3-DMU (Sigma) were assayed by high performance liquid
chromatography (du Souich et al., 1989). In addition, the effect of the
experimental conditions was assessed by measuring P450 content
(spectrally measurable P450) in the hepatocytes (Omura and Sato, 1964).
The amount of proteins in the hepatocytes was determined by the method
of Lowry et al. (1951). The lipid peroxidation induced by the
turpentine-induced inflammation and the experimental conditions was
assessed measuring the amount of malondialdehyde formed in the
hepatocytes by the thiobarbituric acid reaction (Ohkawa et al., 1979).
To ensure that coincubation of HINFLA with
RSINFLA or HSINF for 4 h with theophylline did not distort the results, two control experiments were conducted. First, the rate of formation of
theophylline metabolites was assessed in microsomes from
HCONT or HINFLA
preincubated with RSINFLA or
HSINF for 4 h. To 1 mg of microsomal
proteins in 0.1 M phosphate buffer (pH 7.4) was added NADP (0.5 mM),
glucose 6-phosphate (5 mM), MgCl2 (5 mM), and
glucose-6-phosphate dehydrogenase (2 international units), and
theophylline (176 µM) to a final volume of 1 ml. Following 4 h
of incubation at 37°C, the incubation media was collected and frozen
at 20°C until theophylline, 3-MX, 1-MU, and 1,3-DMU were assayed by
HPLC (n = 3). Second, HCONT and
HINFLA were incubated with
RSINFLA or HSINF for 4 h, and the William's medium E was changed with fresh medium containing
only theophylline (176 µM) and incubated for an additional 4 h
(n = 4). Under both experimental conditions, the
formation of 3-MX, 1-MU, and 1,3-DMU decreased by approximately 35, 30, and 45%, respectively. Simultaneous incubation
of HINFLA, RSINFLA, or
HSINF and theophylline resulted in decreases in
the formation of 3-MX, 1-MU, and 1,3-DMU of the same order.
To demonstrate whether the addition of ROI to cultured hepatocytes
inactivates or reduces the amount of selected apoproteins of the P450,
HCONT (n = 5) and
HINFLA (n = 5) were incubated
with different concentrations of
H2O2 (0.01, 0.05, 0.25, and
1.0 mM) and of sodium nitroprusside (0.01, 0.05, 0.25, and 1.0 mM). The content of P450, formation of theophylline metabolites, and lipid peroxidation were assessed 4 h later. CYP1A1 and -1A2 proteins were measured by Western blot analysis in two samples of
HINFLA incubated with 1.0 mM
H2O2 or sodium
nitroprusside. H2O2 was
selected because of its ability to diffuse into the hepatocytes, act as a source of hydroxyl radicals (·OH), produce cellular damage,
and suppress the transcription of P450 apoproteins (Karuzina and
Archakov, 1994). Sodium nitroprusside was selected because it can
generate NO·, which is able to down-regulate different
apoproteins of the P450 (Stadler et al., 1994).
The role of ROI in the decrease in P450 content and activity induced by
an inflammatory reaction was further investigated by preincubating
HINFLA with an inhibitor of NOS,
N
-nitro-L-arginine
methyl ester (L-NAME; 0.05, 0.25, and 1.0 mM), dimethylthiourea (6.25, 12.5, and 50 mM), and
N-acetylcysteine (0.05, 0.25, and 1.0 mM) (all from Sigma)
for 30 min. Thereafter, 100 µl of RSINFLA
(n = 5) and HSINF
(n = 4) was added to HINFLA, and
P450 content, theophylline metabolites formation, and lipid peroxidation were assessed 4 h later. CYP1A1 and -1A2 proteins were measured by Western blot analysis in samples of hepatocytes incubated with L-NAME (1.0 mM), dimethylthiourea
(50 mM), or N-acetylcysteine (1.0 mM), and
RSINFLA (n = 3) or
HSINF (n = 3). Dimethylthiourea was used, because it is an effective ·OH scavenger able to
penetrate into the cells (Heo et al., 1995), and
N-acetylcysteine, a precursor of glutathione, is able to
react with ROI (Moldeus and Cotgreave, 1994).
To investigate whether the inhibition of antioxidant enzymes potentiate
the decrease in P450 content and activity induced by the inflammatory
reaction, RSINFLA (100 µl, n = 6) and HSINF (100 µl, n = 4)
were preincubated with HINFLA for 30 min with 3-amino-1,2,4-triazole (10, 25, and 100 mM) (Sigma), an inhibitor of
catalase,
DL-buthionine-(S,R)-sulfoximine
(2.5, 6.25, and 25 mM) (Sigma), an inhibitor of glutathione peroxidase,
and diethyldithiocarbamate (1.0, 2.5, and 10 mM) (Sigma), an inhibitor
of superoxide dismutase. P450 content, formation of theophylline
metabolites, and lipid peroxidation were assessed 4 h later.
CYP1A1 and -1A2 proteins were measured by Western blot analysis in
samples of HINFLA incubated with
3-amino-1,2,4-triazole (100 mM),
DL-buthionine-(S,R)-sulfoximine (25 mM), or diethyldithiocarbamate (10 mM), and
RSINFLA (n = 2) or
HSINF (n = 4).
Western Blot Analysis.
Proteins were separated by polyacrylamide gel electrophoresis (7.5%
polyacrylamide) under nonreducing conditions (Smith, 1994). Separated
proteins were electrophoretically transferred to a nitrocellulose membrane using a semidry transfer process (Bio-Rad, Hercules, CA).
CYP1A1 and -1A2 proteins were detected with a polyclonal anti-rabbit
CYP1A1 (Oxford Biochemical Research, Oxford, MI) and visualized with an
alkaline phosphatase-conjugated secondary goat antibody using nitro
blue tetrazolium as the substrate (Kruger, 1994). CYP3A6 protein was
detected with a monoclonal anti-rat CYP3A1 (Oxford Biochemical
Research), using a secondary antibody conjugated to a chemiluminescence
reagent (horseradish peroxidase enzyme), and visualized by
autoradiography (Thorpe et al., 1985). The intensities of the bands
were measured with the software UN-SAN-IT gel version 5.1 (Silk
Scientific, Inc., Orem, UT). .
Statistical Analysis. All results are reported as means ± S.E. The comparison of the results from the various experimental groups and their corresponding controls was carried out using a one-way ANOVA followed by Newman-Keul's post hoc test. The differences were considered significant when P < .05.
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Results |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Effect of Rabbit and Human Serum on P450 Content and Activity. In rabbits with a turpentine-induced inflammatory reaction, seromucoid concentrations were 72.7 ± 2.4 mg/dl compared with 20.8 ± 2.6 mg/dl in control rabbits (P < .05). The content of P450 in HINFLA was lower than in HCONT, i.e., 0.22 ± 0.01 versus 0.34 ± 0.04 nmol/mg of protein (P < .05). By comparison to HCONT, the formation of 3-MX, 1-MU, and 1,3-DMU was reduced by 30, 50, and 28%, respectively, in HINFLA (Table 1). CYP1A1 and -1A2 levels were 43% and 48% lower, respectively, in HINFLA than in HCONT. The amount of CYP3A6 protein was almost undetectable in HINFLA (Fig. 1).
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Effect of H2O2 and Sodium Nitroprusside on
P450 Content and Activity.
Viability of hepatocytes was not affected by concentrations of 
1 mM
H2O2 or sodium
nitroprusside. Exposure of HCONT to various concentrations of H2O2 or
sodium nitroprusside produced a dose-dependent decrease in P450 content
(Fig. 2). As a consequence,
H2O2 and sodium
nitroprusside diminished dose dependently the formation of theophylline
metabolites, 3-MX, 1-MU, and 1,3-DMU (Table 1). Lipid peroxidation, as
expressed in terms of the amount of malondialdehyde generated, was
increased with the addition of
H2O2 and sodium nitroprusside (Fig. 2). Total P450 content, biotransformation of
theophylline, and hepatic lipid peroxidation were not significantly influenced by the lowest concentration of
H2O2 tested (0.01 mM).
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Effect of L-NAME and Antioxidants on Rabbit and Human Serum-Mediated Decrease in P450 Content and Activity. In preliminary studies (n = 5), HINFLA was incubated with L-NAME, dimethylthiourea, or N-acetylcysteine and with serum from control rabbits or healthy humans to assess whether these antioxidants affect P450 content, theophylline metabolism, or lipid peroxidation. The results indicated that at the concentrations equal or lower than 1, 50, and 1 mM, L-NAME, dimethylthiourea, or N-acetylcysteine did not affect the parameters assessed.
L-NAME, dimethylthiourea, or N-acetylcysteine partially prevented the decrease in P450 content and the inhibition of theophylline metabolism, as well as the increase in lipid peroxidation mediated by RSINFLA and HSINF (Figs. 4 and 5, and Tables 2 and 3). At the highest concentration tested, all three compounds elicit a similar protection on the formation of theophylline metabolites, i.e., about 60%. The amount of CYP1A1 and -1A2 proteins in HINFLA were not modified by the presence of L-NAME, dimethylthiourea, or N-acetylcysteine. The relative densities of CYP1A1 in HINFLA and HINFLA in the presence of HSINF, L-NAME, dimethylthiourea, or N-acetylcysteine were 13,860, 13,230, 10,080, 9,200, and 8,820, respectively, and for CYP1A2 these values were 12,600, 13,860, 11,970, 12,560, and 9,454, respectively.
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Effect of Inhibitors of Antioxidant Enzymes on the Decrease of P450 Content and Activity Induced by Rabbit and Human Serum. In preliminary studies (n = 5), HINFLA was incubated with the inhibitors of the antioxidant enzymes and with serum from control rabbits or healthy humans to assess whether at the concentrations used 3-amino-1,2,4-triazole, DL-buthionine-(S,R)-sulfoximine, or diethyldithiocarbamate had a direct effect on P450 content, theophylline metabolism, or lipid peroxidation. The results indicated that, at the concentrations used, none of the inhibitors of the antioxidant enzymes affected the parameters assessed.
3-Amino-1,2,4-triazole and diethyldithiocarbamate significantly potentiated the reduction in P450 content, the inhibition of theophylline metabolism, as well as the increase in lipid peroxidation induced by RSINFLA or HSINF in a dose-dependent manner (Figs. 6 and 7). DL-Buthionine-(S,R)-sulfoximine (25 mM) potentiated the inhibition of 1,3-DMU formation by both sera (Tables 4 and 5) and potentiated the increase in lipid peroxidation mediated by HSINF. The addition of 3-amino-1,2,4-triazole, DL-buthionine-(S,R)-sulfoximine, or diethyldithiocarbamate to hepatocytes cultured with RSINFLA did not change the amounts of CYP1A1 or -1A2 proteins. In the presence of HSINF, 3-amino-1,2,4-triazole and DL-buthionine-(S,R)-sulfoximine did not change the amounts of CYP1A1 or -1A2 proteins, but diethyldithiocarbamate decreased the amounts of CYP1A1 and -1A2 proteins to half the control values. The relative densities of CYP1A1 in HINFLA alone, and HINFLA incubated with HSINF, 3-amino-1,2,4-triazole, and DL-buthionine-(S,R)-sulfoximine and diethyldithiocarbamate were 13,230, 11,760, 10,290, 10,290, and 4,410, respectively, and for CYP1A2, these values were 18,375, 16,170, 13,965, 13,965, and 4,410, respectively.
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Discussion |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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The present results confirm that, in vivo in the rabbit, a
turpentine-induced inflammatory reaction down-regulates hepatic CYP1A1,
-1A2, and -3A6 proteins and show that the incubation of RSINFLA or HSINF with
HINFLA for 4 h reduces P450 content and the
formation of theophylline metabolites without decreasing the amounts of
CYP1A1 and -1A2 proteins. To explain this apparent contradiction,
several elements should be taken into account. First, the
spectrophotometric measure of P450 is based on the fact that CO binds
to reduced iron (Fe2+) of the heme moiety at the
binding site of O2. Therefore, binding of CO to
Fe2+, and spectrophotometric measure of P450,
depends upon the availability of this binding site. On the other hand,
catalytic activity of P450 isoforms also depends upon this binding site
to form the P450-dioxygen complex necessary to transfer an oxygen atom
to the substrate. Immunoquantitation by using an antibody to a specific sequence of amino acids will measure the amount of isoforms; however, it does not indicate whether the isoform measured is active. Because the formation of theophylline metabolites is essentially dependent upon
CYP1A1 and -1A2 activity (Kurdi et al., 1999), we may postulate that
the decrease in the rate of theophylline metabolism is due to a
decrease in CYP1A1 and/or -1A2 activity, because their amounts are not
modified. Supporting such hypothesis, spectrally measurable P450 in
HINFLA was reduced by
RSINFLA or HSINF.
Sodium nitroprusside, a source of NO·, reduces P450 content as
well as the formation of theophylline metabolites without affecting the
amounts of CYP1A1 and -1A2 proteins, while lipid peroxidation is
increased. Further supporting a role for NO· in the decrease of
spectrally measurable P450 content and activity is the fact that the
addition of L-NAME to the incubation media partially
prevents RSINFLA- or
HSINF-induced diminution in P450 content and
activity. NO· binds to the same binding site on the reduced iron
of the heme as do CO and O2, and as a
consequence, NO· decreases spectrally measurable P450, as well
as its activity (Khatsenko et al., 1993; Wink et al., 1993). In
agreement with the present results, it has been shown that NO·
is able to decrease the activity CYP1A1, -1A2, -2B1, -2B2, -2C11, and
-3A2 (Khatsenko et al., 1993; Wink et al., 1993; Stadler et al., 1994).
Moreover, NO· can inactivate CYP2E1, and the inactivated form
retains the epitope for its recognition when assayed by Western blot
analysis (Gergel et al., 1997). Therefore, in the absence of changes in
the amount of CYP1A1 and -1A2 proteins, and in agreement with the
conclusions reached by Khatsenko et al. (1993), we propose that, under
the present experimental conditions, RSINFLA and
HSINF induce the release of NO·, which
operates inactivating CYP1A1 and -1A2.
N-Acetylcysteine attenuates the decrease in P450 content and
activity induced by the addition of RSINFLA or
HSINF, and 3-amino-1,2,4-triazole, an inhibitor
of catalase (Mian and Martin, 1995) potentiates the effect of
RSINFLA or HSINF on P450
content and activity, without affecting CYP1A1 and -1A2. Exposure of
HCONT or HINFLA to various concentrations of H2O2 for
4 h reduces P450 content as well as the formation of theophylline
metabolites without affecting the amounts of CYP1A1 and -1A2 proteins,
strongly supporting that H2O2 is contributing to
RSINFLA- or HSINF-induced
decrease in P450 content and activity. The possibility that
H2O2 might directly decrease the activity of CYP1A1 was already proposed some years ago
(Flowers and Miles, 1991). The exact mechanism by which
H2O2 would decrease the
activity of selected P450 isoforms remains unclear, but it has been
shown that H2O2 formed in
the hemoprotein active center can interact with the enzyme-associated
Fe2+ leading to heme destruction and enzyme
inactivation (Karuzina and Archakov, 1994; Archakov et al., 1998). On
the other hand, H2O2 might
simply be a second messenger in the signaling pathway leading to the
inactivation and/or the down-regulation of selected P450 isoforms (Bae
et al., 1997; Lowe et al., 1998).
The fact that
DL-buthionine-(S,R)-sulfoximine, an
inhibitor of glutathione peroxidase (Masaki et al., 1998), does not
potentiate the effect of RSINFLA or
HSINF suggests that the effect of
N-acetylcysteine, a source of reduced glutathione the
substrate of glutathione peroxidase (Singh et al., 1998), is due to its
ability to scavenge H2O2
(Vanderbist et al., 1996). On the other hand, diethyldithiocarbamate,
an inhibitor of superoxide dismutase (Martin et al., 1994), potentiates
RSINFLA- or HSINF-mediated
reduction in P450 content and activity, without affecting the amounts
of CYP1A1 and -1A2 proteins. Superoxide dismutase transforms superoxide
(O
2) to H2O2,
which is catalyzed to water by catalase and glutathione peroxidase
(Southorn and Powis, 1988). Because it has been shown that O2
is able to reduce the activity of CYP1A1 (Flowers and Miles, 1991), we
postulate that O2 has contributed directly in the decrease
in P450 activity, or indirectly as a source of
H2O2. Despite the fact that
dimethylthiourea is a rather specific scavenger of ·OH, a role
for ·OH is less clear, because dimethylthiourea can also
scavenge peroxynitrite (Hara et al., 1998). Nevertheless, the present
results suggest that several species of ROI are implicated in the
RSINFLA- or HSINF-mediated
reduction in P450 content and activity. This multiplicity of ROI may
explain why L-NAME and other antioxidants confer
only a partial protection to the serum-mediated P450 decrease in activity.
Alternatively, ROI may have reduced the activity of selected isoforms
of the P450 indirectly by inducing the phosphorylation of the isoforms.
Effectively, activation of kinases can phosphorylate P450 resulting in
its inactivation (Rhee, 1999). In vitro, several isoforms of rabbit and
rat P450 can be phosphorylated on a serine residue at positions
127-129 by cAMP-dependent kinase A and protein kinase C (Moritz et
al., 1998). ROI, specifically O2 and
H2O2, appear to play an
important role as messengers as well as stimulators of protein tyrosine
kinase (Bae et al., 1997; Lowe et al., 1998), protein kinase C (Boyer
et al., 1995), protein kinase A (Suzuki et al., 1997), and
mitogen-activated protein kinases (Goldstone and Hunt, 1997). The
mechanism by which phosphorylation inactivates P450 is unclear; it has
been shown that phosphorylation increases the uncoupling from
NADPH-dependent hydroxylation of xenobiotics by P450, P450-reductase,
and cytochrome b5 (Mkrtchian and Andersson, 1990). Indeed, further studies are required to understand the signal
transduction pathways activated by RSSINF and
HSINF leading to P450 inactivation.
In this laboratory we have shown that the decrease in P450 activity
elicited by RSINFLA and
HSINF is mediated by interleukin-6 (IL-6), and by
IFN-, IL-1
, and IL-6, respectively (Bleau et al., 2000). Keeping
in mind the diversity of serum mediators, multiple sources of ROI are
possible: membrane and cytosolic NADPH oxidase, xanthine oxidase, the
mitochondrial respiratory chain, and the P450 system. Concerning the
NADPH oxidase system, IL-6, IL-1 and IFN- can activate
phospholipase A2 (McPhail et al., 1993) to produce arachidonic acid,
which will be transformed into leukotrienes by 5-lipoxygenase and
generate ROI (Bonizzi et al., 1999). Alternatively, both arachidonic
acid and leukotrienes may activate membrane or cytosolic NADPH oxidase,
an important source of ROI (Rhee 1999). Cytokines such as IFN-,
IL-1, and IL-6 are capable to activate xanthine oxidase to generate
O2 (Ghezzi et al., 1985). Supporting that xanthine oxidase is
a source of ROI, turpentine-induced inflammatory reaction increases
hepatic xanthine oxidase activity (Proulx and du Souich, 1995). In
response to multiple stimuli, such as sepsis, cytokines, or ceramide,
Complex I to III of the mitochondrial respiratory chain will generate ROI, which may be used as signal transduction messengers to activate transcription factors, the expression of manganese superoxide dismutase, nitric oxide synthase, and/or lead to apoptosis (Degli Esposti and McLennan, 1998). Finally, in the hepatocyte, several P450
isoforms are able to generate ROI (Serino et al., 1993), specifically,
CYP2E1 may generate H2O2
(Gergel et al., 1997); CYP3A4, -1A1, -1A2, and -2B6 produce O2
(Puntarulo and Cederbaum, 1998); and CYP3A yields NO· (Kuo et
al., 1995).
In conclusion, 48 h after a turpentine-induced acute inflammatory
reaction there is a down-regulation of CYP1A1, -1A2, and -3A6 proteins.
When incubated for 4 h with HINFLA, the
serum of rabbits with a turpentine-induced inflammatory reaction and
the serum of humans with a viral infection decrease spectrally
measurable P450 and the formation of theophylline metabolites without
affecting the amounts of CYP1A1 and -1A2 proteins. This suggests that a short incubation of HINFLA with serum decreases
the activity of CYP1A1 and -1A2. The fact that L-NAME and
antioxidants are unable to completely abrogate the serum-induced
decrease in activity of P450 indicates that several species of ROI
contribute to the decrease in activity of hepatic P450, possibly
NO·, H2O2, and
O2.
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Acknowledgments |
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The authors are grateful to Mrs. Lucie Héroux for her skilful technical assistance. A.O.S El-Kadi is recipient of a scholarship from Novartis Pharma Inc. (Canada).
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Footnotes |
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Received November 19, 1999; accepted May 26, 2000.
This study was supported by the Medical Research Council of Canada (Grant MT-14478).
Send reprint requests to: Patrick du Souich, MD, PhD, Department of Pharmacology, Faculty of Medicine, University of Montréal, P.O. Box 6128, Stat. Centre-Ville, Montréal, Québec, Canada H3C 3J7. E-mail: patrick.du.souich{at}umontreal.ca
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Abbreviations |
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Abbreviations used are:
P450, cytochrome P450;
CYP, apoprotein of the cytochrome P450;
1,3-DMU, 1,3-dimethyluric acid;
HCONT, hepatocytes from control rabbits;
HINFLA, hepatocytes from rabbits with a turpentine-induced
acute inflammatory reaction;
HSINF, serum of humans with a
viral infection;
IFN-, interferon-;
IL-, interleukin;
L-NAME, N-nitro-L-arginine methyl
ester;
1-MU, 1-methyluric acid;
3-MX, 3-methylxanthine;
NO·, nitric oxide radical;
NOS, nitric oxide synthase;
·OH, hydroxyl
radical;
O2, superoxide radical;
ROI, reactive oxygen
intermediates;
RSINFLA, serum from rabbits with a
turpentine-induced acute inflammatory reaction.
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References |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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requires 5-lipoxygenase or NADPH oxidase activity.
Mol Cell Biol
19:
1950-1960-Con1 on hepatic cytochrome P-450 and protein synthesis and degradation in hepatic microsomes.
Int J Immunopharmacol
13:
903-912[Medline]. and endotoxin in primary rat hepatocytes.
Biochem Pharmacol
54:
729-737[Medline].This article has been cited by other articles:
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