Introduction

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In human, viral and bacterial infections as well as influenza and BCG vaccinations decrease the clearance of exogenous substances such as theophylline and antipyrine, secondary to a decrease in activity of multiple isoforms of the cytochrome P450 (P450¹) (Morgan, 1997). As a consequence, bacterial and viral inflammatory reactions are cause of severe drug toxicity, essentially in pediatric and geriatric populations (Hendeles et al., 1977; Ziment, 1982; Koren and Greenwald, 1985). In animal models, infectious and noninfectious acute inflammatory reactions, such as those induced by endotoxin and turpentine, also diminish the rate of metabolism of xenobiotics (Parent et al., 1992; Morgan, 1997).

In response to viral infections, the concentration in blood of many cytokines is increased (Ramshaw et al., 1997). It has been assumed that in vivo cytokines are responsible for P450 depression because in vitro cytokines can depress multiple hepatic P450 isoforms and their mRNAs. For instance, INF- depresses CYP1A2, 2A6, 2B6, and 3A4; IL-6 depresses CYP1A1, 1A2, 2D, 3A4, and 4A1; and IL-1 down-regulates 1A2, 2C11, 2D6, 2E1, and 3A (Fukuda et al., 1992; Trautwein et al., 1992; Donato et al., 1997; Parmentier et al., 1997). Furthermore, it has been reported that IFN- (Stanley et al., 1991), IL-1 (Peterson and Renton, 1986), and TNF- (Paton and Renton, 1998) can also reduce the activity of several P450 isoforms.

In vitro, following an incubation period of 4 h with hepatocytes, serum from humans with an upper respiratory tract viral infection (HS_URVI) and serum from rabbits with a turpentine-induced acute inflammatory reaction (RS_TIAR) decreases the activity of CYP1A1 and 1A2 without affecting the amount of these proteins (El-Kadi et al., 1997). The mediators responsible for the decrease in P450 activity are IFN-, IL-6, and IL-1 in HS_URVI, and IL-6 in RS_TIAR (Bleau et al., 2000), demonstrating that in vivo, infectious, and noninfectious inflammatory reactions generate serum mediators, namely, cytokines, capable to reduce P450 activity.

Since in vivo, an inflammatory reaction depresses the expression of multiple isoforms of the P450, it was of interest to establish whether HS_URVI and RS_TIAR are capable to down-regulate the expression of selected P450 isoforms in hepatocytes. Specifically, the aims of the present study were 1) to assess the effect of RS_TIAR and HS_URVI on the activity and expression of hepatic CYP1A1, 1A2, and 3A6 following 24-h incubation periods; and 2) to compare the differences in effect between RS_TIAR and HS_URVI as a function of the source of the hepatocytes, i.e., hepatocytes harvested from control rabbits (H_CONT) and hepatocytes from rabbits with a TIAR (H_INFLA). The use of H_INFLA was justified because the density of surface receptors to cytokines is greater than in H_CONT (Dinarello, 1994). Theophylline was used to assess the activity of some isoforms of the P450. In the rabbit, theophylline is primarily metabolized by CYP1A2 and CYP1A1, and to a minor degree by CYP3A6; CYP1A1, and CYP1A2 both contribute to the formation of theophylline three metabolites 3-methylxanthine (3 MX), 1-methyluric acid (1 MU), and 1,3-dimethyluric acid (1,3DMU) (Kurdi et al., 1999).

	Materials and Methods

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Collection of Hepatocytes and Serum from Rabbits. Male New Zealand White rabbits (1.8-2.2 kg) were obtained from Ferme Charles Rivers (St-Constant, Québec, Canada). A local inflammatory reaction was induced by the s.c. injection of 5 ml of turpentine (Recochem, Montréal, Québec) distributed into four distinct sites of the back of the rabbits. Forty-eight hours later, blood (20 ml) was withdrawn from the central artery of an ear of the rabbits, controls and with a TIAR; the rabbits were anesthetized, and hepatocytes were isolated. Blood samples were allowed to clot at room temperature for 2 h, centrifuged at 2500 rpm for 5 min, and the serum was decanted and stored frozen at 20°C in 1-ml aliquots until use. The severity of the inflammatory reaction was assessed by measuring serum concentrations of seromucoids (Parent et al., 1992). All the experiments were conducted according to the Canadian Council on Animal Care guidelines for use of laboratory animals.

Collection Serum from Humans. Blood (10 ml) was withdrawn from humans (n = 8) with an inflammatory reaction secondary to an upper respiratory viral infection, at the apex of clinical symptomatology, i.e., 24 h after the appearance of overt manifestations of an upper respiratory tract viral infection, such as rhinorrhea, sneezing, nasal congestion, sore throat, cough, and systemic signs of malaise, including fever, in absence of purulent secretions. Blood samples were allowed to clot at room temperature for 2 h, centrifuged at 2500 rpm for 5 min, and the serum was decanted and stored frozen at 20°C in 1-ml aliquots until use.

Cytochrome P450 Content and Activity. The efficacy of the serum to reduce hepatic P450 content was tested by incubating 200 µl of serum with 2 ml/well of H_CONT and H_INFLA for 4 and 24 h. Hepatic P450 content, evaluated by its ability to bind carbon monoxide, was measured spectrophotometrically as described by Omura and Sato (1964). Protein content in hepatocytes was measured by the method of Lowry et al. (1951).

Western Blot Analysis. Proteins were separated by SDS-polyacrylamide gel electrophoresis (7.5% polyacrylamide) under nonreducing conditions (Smith, 1994). 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, 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 and a horseradish peroxidase-conjugated secondary antibody; chemiluminescence was visualized by autoradiography (Thorpe et al., 1985). The intensities of the bands were measured with a software Un-Scan-It-Gel (Silk Scientific Inc., Orem, UT).

Drugs and Chemicals. The Percoll gradient, Williams' medium E, calf serum, type I rat tail collagen, NaCl, KCl, KH₂PO₄, HEPES, EGTA, glucose, theophylline, 3 MX, 1 MU, and 1,3DMU were purchased from Sigma (St. Louis, MO) and insulin was from Roche Molecular Biochemicals (Mannheim, Germany).

Statistical Analysis. All data 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 analysis of variance followed by Newman-Keuls post hoc test. The effect of RS_TIAR and HS_URVI on the amount of CYP1A1, 1A2, and 3A6 (densitometry values) was compared with the effect of RS_CONT and HS_CONT by using a paired t test. The differences were considered significant when p < 0.05.

	Results

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Effect of a TIAR on P450 Content, Activity, and Amount of P450 Apoproteins. Total P450 content in H_CONT was 0.284 ± 0.014 nmol/mg of protein, a value that was reduced to 0.152 ± 0.012 nmol/mg of protein in H_INFLA (p < 0.05). Compared with H_CONT, when theophylline was incubated for 4 h with H_INFLA, the formation of 1,3DMU, 3 MX, and 1 MU was reduced by 43, 85, and 43%, respectively (p < 0.005), reflecting essentially a decrease in CYP1A1 and 1A2 activity (Table 1). After 24 h of incubation, the formation of 1,3DMU, 3 MX, and 1 MU was reduced by a percentage similar to that reported after 4-h incubation (data not shown). Compared with H_CONT, the TIAR diminished the amount of CYP1A1 by 47%, i.e., densitometry values were 391,687 ± 41,243 in H_CONT and 207,492 ± 30,617 in H_INFLA (n = 3, p < 0.05). CYP1A2 densitometry values were 309,098 ± 57,205 in H_CONT and 205,618 ± 35,456 in H_INFLA (n = 3, p > 0.05). The amount of CYP3A6 apoprotein was reduced to an undetectable level (Fig. 1).

View larger version (50K): [in this window] [in a new window]   Fig. 1.   Effect of a turpentine-induced inflammatory reaction on the amount of CYP1A1, 1A2, and 3A6 apoproteins in hepatocytes from control (HCONT) and rabbits with a turpentine-induced inflammatory reaction (HINFLA), 48 h after the injection of turpentine. Numbers indicate densitometry values.

Effect of RS_CONT and RS_TIAR on P450 of H_CONT Following 4 and 24 h of Incubation. Incubation of RS_CONT with H_CONT for 4 h did not modify P450 content and ability to biotransform theophylline (n = 7) (Table 1). Compared with RS_CONT, RS_TIAR did not modify P450 content, and did not reduce the formation of 3 MX, 1 MU, and 1,3DMU (p > 0.05) (Table 1). Neither RS_CONT nor RS_TIAR affected the amount of CYP1A1, 1A2, and 3A6 apoproteins in H_CONT (data not shown).

View larger version (44K): [in this window] [in a new window]   Fig. 2.   Effect of serum from control (RSCONT) and rabbits with a turpentine-induced inflammatory reaction (RSINFLA) on the amount of CYP1A1, 1A2, and 3A6 apoproteins in hepatocytes from control rabbits (HCONT) and rabbits with a turpentine-induced inflammatory reaction (HINFLA), after a 24-h period of incubation. Numbers indicate densitometry values.

Effect of RS_CONT and RS_TIAR on P450 of H_INFLA Following 4 and 24 h of Incubation. Following 4 h of incubation, and compared with RS_CONT, RS_TIAR did not reduce P450 content in H_INFLA, but reduced the formation of 1 MU and 1,3DMU by 34 and 26%, respectively (p < 0.005, n = 7) (Table 2). On the other hand, the amount of CYP1A1 and 1A2 proteins was not affected in H_INFLA (data not shown).

In Vivo Effect of an URVI on Metabolism of Theophylline in Human Volunteers. During the symptom-free period in five control volunteers, the 24-h urinary recovery of theophylline and its metabolites accounted for 69% of the dose administered, of which 13.3% was 3 MX, 20.3% 1 MU, and 52.5% was 1,3DMU. While the volunteers presented symptoms of URVI, the 24-h urinary recovery of theophylline and its metabolites decreased to 58% (p < 0.05). Compared with the control period, recovery of 3 MX, 1 MU, and 1,3DMU was decreased by 30, 29, and 14% (p < 0.05), respectively (Fig. 3).

View larger version (48K): [in this window] [in a new window]   Fig. 3.   Amount of 3 MX, 1 MU, and 1,3DMU recovered in a 24-h urinary collection from five volunteers with and without an upper respiratory viral infection after oral intake of 300 mg of theophylline.

Effect of HS_CONT and HS_URVI on P450 of H_CONT Following 4 and 24 h of Incubation. Incubation of HS_CONT with H_CONT for 4 and 24 h did not change hepatic P450. In contrast, following 4 h of incubation, HS_URVI did not modify total P450 content but decreased the formation of 1 MU and 1,3DMU by 28 and 32%, respectively (p < 0.05) (Table 3). The amounts of CYP1A1, 1A2, and 3A6 were not affected (data not shown).

View larger version (43K): [in this window] [in a new window]   Fig. 4.   Effect of serum from volunteers with (HSURVI) and without (HSCONT) an upper respiratory tract viral infection reaction on the amount of CYP1A1, 1A2, and 3A6 apoproteins in hepatocytes from control rabbits (HCONT) and rabbits with a turpentine-induced inflammatory reaction (HINFLA), after a 24-h period of incubation. Numbers indicate densitometry values.

Effect of HS_CONT and HS_URVI on P450 of H_INFLA Following 4 and 24 h of Incubation. Incubation of HS_URVI with H_INFLA for 4 h decreased total P450 content from 0.140 ± 0.013 nmol/mg of protein to 0.101 ± 0.011 nmol/mg of protein (p < 0.05). HS_URVI lowered the concentration of 1 MU and 1,3DMU by 42 and 32%, respectively (p < 0.05) (Table 3), without affecting the amount of CYP1A1 and 1A2 (data not shown).

	Discussion

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The present study demonstrates that RS_TIAR does not affect P450 amount or activity when incubated for 4 h with H_CONT, however, it decreases P450 activity in H_INFLA. When RS_TIAR is incubated with H_CONT for 24 h, P450 activity is reduced, as well as the amount of CYP3A6 protein. Incubation of RS_TIAR with H_INFLA for 24 h decreases total P450 content and activity, without affecting CYP1A1 or 1A2. Keeping in mind that theophylline is primarily biotransformed by CYP1A2 (Kurdi et al., 1999), these results show that RS_TIAR elicits a dual effect on P450: it reduces the activity of CYP1A1 and 1A2, and down-regulates CYP3A6. RS_TIAR effects on P450 depend upon the length of incubation, and the state of the hepatocytes, i.e., control or primed.

The RS_TIAR-induced decrease in P450 activity could be a general phenomenon since the down-regulation of P450 isoforms by lipopolysaccharides is preceded by their inactivation (Sewer et al., 1998). Nitric oxide may have a pivotal role in P450 inactivation by both, the turpentine induced inflammation (El-Kadi et al., 2000), and lipopolysaccharide-induced endotoxemia (Sewer et al., 1998; Takemura et al., 1999). The fact that the biotransformation of theophylline and total P450 content in H_INFLA are reduced by RS_TIAR without changes in the amount of CYP1A1, 1A2, and 3A6 proteins, further supports that nitric oxide contributes to the reduction in P450 activity. As discussed, upon binding to Fe²⁺ and Fe³⁺-heme, nitric oxide impedes O₂ binding and as a consequence, it reduces theophylline biotransformation. In parallel, nitric oxide binding to Fe²⁺-heme impedes the binding of carbon monoxide used for the spectrophotometric determination of total P450 content (Omura and Sato, 1964), resulting in the apparent decrease in total P450 content.

Following 24-h incubation with H_CONT, RS_TIAR reduction of P450 activity and CYP3A6 expression may be explained by the fact that IL-6 is the primary serum mediator induced by the TIAR (Bleau et al., 2000; Siewert et al., 2000). IL-6 preferentially depresses activity and expression of CYP3A, with little effect on the expression of CYP1A1 and 1A2 proteins (Muntané-Relat et al., 1995). CYP3A6 down-regulation by IL-6 has been associated with the decrease in the expression of pregnane X receptor and constitutively activated receptor mRNAs (Pascussi et al., 2000). On the other hand, it has been shown that IL-6 increases the expression of inducible nitric-oxide synthase (NOS2) (Ma and Zhu, 2000). Since CYP3A6 in H_INFLA is already depressed by the TIAR, incubation of H_INFLA with RS_TIAR for 24 h reduces only CYP1A1 and 1A2 activity.

When HS_URVI is incubated for 4 h with H_CONT, there is a decrease in P450 activity without changes in CYP1A1, 1A2, or 3A6 apoproteins; when the incubation is prolonged for 24 h, the decrease in P450 activity is accompanied by a reduction in the amount of CYP3A6. Incubation of HS_URVI with H_INFLA for 4 h reduces activity and total P450 content, and incubation for 24 h reduces activity, total P450 content and amount of CYP1A1 and 1A2 proteins. These experiments clearly indicate that P450 down-regulation is preceded by a decrease in activity of several P450 isoforms. As demonstrated by the in vivo studies in human volunteers, the potency of HS_URVI to reduce P450 activity is directly associated with the repercussions of the viral infection on the in vivo metabolism of theophylline.

There is evidence pointing out that following the incubation of HS_URVI with H_CONT and H_INFLA for 4 h, the decrease in P450 activity is also mediated by nitric oxide (El-Kadi et al., 2000). The decrease in P450 activity in H_CONT following 4-h incubation with HS_URVI implies that the source of nitric oxide may not be NOS2, since longer periods are required to increase its expression. A potential source of nitric oxide in H_CONT could be CYP3A6, since this isoform is a direct source of nitric oxide (Servent et al., 1989; Boucher et al., 1992). On the other hand, in H_INFLA after 4 h of incubation, the effect of HS_URVI is almost totally prevented by N-nitro-L-arginine methyl ester (El-Kadi et al., 2000), supporting that nitric oxide is also responsible for the decrease in P450 activity. The source of nitric oxide in H_INFLA could be NOS2, because CYP3A6 is almost completely down-regulated.

Following 24 h of incubation, HS_URVI down-regulates CYP1A1/2 in H_INFLA but not in H_CONT, where only CYP3A6 is down-regulated. Possibly several factors associated to the density of surface receptors, the kind of cytokines stimulated by influenza, and the ability of these cytokines to depress selective P450 isoforms contribute to explain these differences. In H_INFLA primed by the TIAR, the density of surface receptors to IFN-, IL-1, TNF-, and IL-6 could be increased, since not only inflammatory stimuli increase the density of surface receptors but also higher levels of circulating cytokines produce the same effect (Volpes et al., 1991; Dinarello, 1994). In humans, influenza increases the concentration of cytokines known to down-regulate P450 isoforms, such as IL-1, IL-6, TNF-, and IFN- (Han and Meydani, 2000). The ability of these cytokines to depress CYP3A isoforms is greater than that to down-regulate CYP1A1/2 (Abdel-Razzak et al., 1993; Muntané-Relat et al., 1995). These differences may be explained in part by the mechanism through which a cytokine depresses the expression of an isoform. For example, IL-1 promotes a pretranscriptional repression, while IFN- exerts a post-transcriptional suppressive effect on CYP3A6 expression (Calleja et al., 1998), and a pretranscriptional down-regulation of CYP1A2 (Abdel-Razzak et al., 1993), however, its effect is weak compared with IL-1 (Calleja et al., 1997). IL-6 is a stronger pretranscriptional repressor of CYP3A4 mRNA than IL-1 and TNF-, but TNF- appears to elicit a stronger pretranscriptional effect on CYP1A1/2 than IL-1 and IL-6 (Muntané-Relat et al., 1995). We may postulate that CYP3A6 expression is more sensitive to the effect of cytokines because IL-1, IL-6, TNF-, and IFN- down-regulate CYP3A6 expression through pre- and post-transcriptional mechanisms, and CYP1A1/2 only by a pretranscriptional repression mechanism.

In conclusion, the present study demonstrates 1) that the effect of RS_TIAR and HS_URVI depends upon the susceptibility of the hepatocyte (H_CONT or primed H_INFLA), emphasizing the importance of the model for an accurate interpretation of the results; 2) that P450 down-regulation is preceded by a decrease in P450 activity; 3) that the differences between RS_TIAR and HS_URVI are primarily dependent upon the mediators contained in the serum, indicating that the nature of the inflammatory reaction is of importance to determine the repercussions on P450 activity and expression; and 4) the present results confirm that CYP3A6 is more vulnerable than CYP1A1 and 1A2 to the down-regulation provoked by an inflammatory challenge, a fact that may have practical consequences when it is taken into account the relevancy of this isoform in drug metabolism.