Introduction

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Cytochrome P450 is a superfamily of enzymes that is well known for their role in the metabolism and excretion of drugs from the body. These enzymes are primarily, but not exclusively, found in the liver and adrenal glands, and they are also distributed throughout the body in diverse areas, such as the intestines, skin, lungs, and brain (Norris et al., 1996). Cytochrome P450 enzymes in the brain are approximately 0.5 to 3% of the content in liver and should not significantly contribute to overall drug elimination. These enzymes, however, have been shown to be highly localized in discrete areas within the brain parenchyma and, thus, may alter the local actions or concentrations of neuroactive drugs (Majewska et al., 1986). In addition, it has been shown that cytochrome P450 in the brain may have homeostatic functions because isoforms have been shown to participate in cerebral blood vessel tone and also in the synthesis of neuroactive steroids (Walther et al., 1987; Warner et al., 1994; Harder et al., 1997). Current evidence shows that CYP1A1/2, CYP2B1, CYP2E1, CYP2D1, novel forms from the CYP3A and CYP4F families, and CYP7B exist in the brain and are regionally located in both neuronal and glial cells (Strobel et al., 1995; Miksys et al., 2000).

Recent evidence from our laboratory and others has shown that several cytochrome P450 isoforms found in the brain are depressed during a localized CNS¹ inflammatory response induced by an i.c.v. injection of lipopolysaccharide (LPS) (Shimamoto et al., 1998; Renton et al., 1999; Renton and Nicholson, 2000; Nicholson and Renton, 2001). The mechanism by which LPS-evoked inflammation causes a depression of CYP1A activity has not been elucidated, although it is likely to involve mediators generated from immunocompetent cells within the brain parenchyma during inflammation (Montero-Menei et al., 1996). In response to an immune stimulus, glial cells, specifically astrocytes and microglia, become activated and stimulate the acute phase response. This process is characterized, in part, by the release of immune mediators including cytokines, prostanoid products, and nitric oxide (NO) (Rivest et al., 2000).

In a previous report from this laboratory, we demonstrated that glial cells harvested from rat brain tissue are predominately astrocytes (approximately 95%), and it is these astrocytes that are capable of expressing the cytochrome P450 isoforms CYP1A1/2 in response to the chemical inducer dibenz[a,h]anthracene (DBA). Cultured astrocytes have been used as an in vitro model for assessing the local effects of immune modulators on cytochrome P450 activity in CNS-derived cells (Tindberg et al., 1996; Nicholson and Renton, 1999). In this article, we examined the role of cytokines in mediating the down-regulation of cytochrome P450 in CNS-derived cells.

	Materials and Methods

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Reagents. Rat TNF- and IL-1 were purchased from Cedarlane Labs, Inc. (Hornby, ON, Canada), and IL-1, IL-6, and IFN- were obtained from R&D Systems (Minneapolis, MN). DBA, Escherichia coli lipopolysaccharide (serotype, 0127:B8), and all other reagents were purchased from Sigma-Aldrich Chemical Co. (St. Louis, MO) except those noted below.

Isolation and Treatment of Astrocytes. Astrocytes were isolated from newborn Sprague-Dawley rats obtained from Charles River Labs (Montreal, QC, Canada), using a method described by Nicholson and Renton (1999). Each culture was derived from cells pooled from 12 to 16 litter mates. After a 2-week incubation period, cultures were approximately 90 to 95% confluent and contained predominantly astrocytes, as determined using antibodies directed against the astrocyte marker glial fibrillary acid protein with a minor contribution of microglia and oligodendrocytes (3 and 2%, respectively) (Hertz et al., 1989). At this time, fresh serum-free Dulbecco's modified Eagle's medium, containing antibiotic-antimycotic (100 U of penicillin, 100 µg of streptomycin, and 0.25 µg/ml amphotericin B), was added to the cells along with 50 nM DBA to induce CYP1A activity and 50 µl of the drug of interest. Cells were incubated for a further 24-h period before enzymatic assessment. Throughout this article, each experiment is reported as a complete treatment protocol in a single pooled cell preparation and is representative of experiments replicated several times in different cell preparations. Although the values obtained for control treatments differed in replicate experiments, the various treatment protocols produced consistent alterations in the measured parameters relative to these controls.

7-Ethyoxyresorufin O-Dealkylase Activity and Protein Determination. CYP1A activity was determined by measuring the rate of formation of resorufin from ethoxyresorufin using a modification of a procedure described by Burke et al. (1985). Briefly, cells were incubated with 0.6 µM ethoxyresorufin in 5 ml of serum-free culture medium for 2 h at 37°C, then 2 ml of medium was removed, and the fluorescence measured (_ex, 510 nm; _em, 586 nm). CYP1A activity was expressed as the amount (picomoles) of resorufin formed per milligram protein per minute.

Cytokine Determination. After administration of LPS, levels of TNF-, IL-1, and IFN- were measured using Quantikine cytokine assay kits (R&D Systems). The procedure was followed as outlined in the manufacturers' instructions. Media samples were taken from treated cultures at 2, 4, 6, 12, and 24 h after drug addition. Samples were determined in duplicate, and the absorbance was read at 450 nm. Levels of cytokines are reported as the average concentration (nanograms per milliliter) per treatment at each time point. The limit of detection for each cytokine assay was 5 pg/ml for TNF- and IL-1 and 10 pg/ml for IFN-.

Nitric Oxide Determination. Nitric oxide content was measured in culture medium by determining the amount of nitrite (NO₂) formed by reacting equal volumes of culture supernatant and Griess reagent (Spitzer, 1994). The resulting mixture was incubated at room temperature for 15 min, and the absorbance was read at 550 nm. The amount of nitrite formed was expressed as nanomoles of NO₂ per milligram of protein.

Statistical Analysis. All data are reported as the mean ± S.E.M. Multiple comparisons were made using one-way analysis of variance with statistical significance set at p < 0.05 and assessed by the Student-Newman-Keuls test.

	Results

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Effect of LPS on Induced CYP1A Activity. The addition of LPS (25 µg/ml) to cells for a 24-h period resulted in a complete loss of CYP1A activity, as indicated by EROD activity (Fig. 1). When dexamethasone (DEX) (40 µg/ml) was added to cells concurrently with LPS, the decrease in EROD activity was only 28%. This observed decrease was identical to the depression occurring in cells treated with DEX alone.

View larger version (11K): [in this window] [in a new window]   Fig. 1.   The effect of DEX on the LPS-induced decrease in CYP1A activity in cultured astrocytes. Cells were pretreated with a 40-µg/ml dose of DEX for 30 min before the addition of 50 nM DBA and 25 µg/ml LPS. Astrocytes were incubated with drug for 24 h, and then EROD activity was measured (n = 4 plates/treatment). *, significantly different with respect to saline control; #, significantly different with respect to LPS-treated.

LPS-Induced Cytokine Release in Cultured Astrocytes. After the incubation of astrocytes with 25 µg/ml LPS, samples of culture medium were obtained at 2, 4, 6, 12, and 24 h to assess levels of cytokine release during the culture period. These cells responded to LPS by the production of TNF- within 2 h, which remained elevated throughout the culture period (Fig. 2A). Similarly, LPS-treated cells produced IL-1 within 4 h, and the levels were sustained throughout the 24-h period (Fig. 2B). No detectable TNF- or IL-1 was produced in cells treated with an equivalent volume of saline. The addition of DEX to the cultures significantly prevented the production of TNF- and IL-1 stimulated by LPS (Fig. 2, A and B). The levels of cytokines released from cells treated with LPS and DEX were identical to levels observed in cells treated with DEX only. In contrast to TNF- and IL-1, LPS was unable to induce IFN- release in any of the cell treatments at any time point (data not shown).

View larger version (15K): [in this window] [in a new window]   Fig. 2.   LPS induces release of cytokines from cultured astrocytes. Samples of media were taken from plates at 2, 4, 6, 12, and 24 h after LPS exposure. TNF- concentration levels (A) and IL-1 levels (B) were determined in culture medium from these treated cells. Each determination was done in duplicate, and the values were averaged. Media samples were taken from different replicates of the same treatment for each time period studied. , saline;   , LPS; , LPS + Dex;   , Dex.

Cytokines Modulate CYP1A Activity in Astrocytes. Several acute phase cytokines were assessed for their ability to modulate CYP1A activity when added directly to cultured astrocytes (Fig. 3). At a concentration of 20 ng/ml, TNF- caused a 38% decrease in EROD activity (control activity levels 5.01 ± 0.49 pmol of resorufin/mg of protein/min). Incubation of astrocytes with IL-1 lowered EROD activity by 23% at a concentration of 15 ng/ml (control activity level, 2.74 ± 0.11 pmol of resorufin/mg of protein/min). In contrast, IL-6 (28 ng/ml) and IL-1 (9 ng/ml) had no effect on EROD activity when added to astrocytes cultures (control activity levels for IL-6 and IL-1 are 3.12 ± 0.52 and 1.38 ± 0.14 pmol of resorufin/mg of protein/min, respectively) (Fig. 3).

View larger version (13K): [in this window] [in a new window]   Fig. 3.   The effect of cytokines on CYP1A activity in cultured astrocytes. In each case, the solid bars represent the control levels of EROD activity and the hollow bars the activity seen in cytokine-treated cultures. Astrocytes were incubated for 24 h with various concentrations of cytokines (TNF-, 5-20 ng/ml; IL-1, 5-15 ng/ml; IL-6, 14-28 ng/ml; IL-1, 10-15 ng/ml) before the determination of EROD activity. The data illustrated represent the highest concentration of cytokine used in each case. Each treatment represents a separate experiment (n = 4 plates/treatment). *, significantly different from control.

View larger version (8K): [in this window] [in a new window]   Fig. 4.   The effect of IFN- on CYP1A activity in astrocytes. Cells were treated with either 2 or 8 ng/ml IFN- for 24 h, and then the EROD activity was assessed (n = 4 plates/treatment). *, significantly different with respect to control.

Nitric Oxide Release Induced by Immunostimulants. NO levels were determined in a culture medium of cells treated with LPS or cytokines. As shown in Table 1, LPS induced the release of substantial amounts of NO, whereas the cytokines TNF- and IFN- did not release detectable amounts of NO. In the case of IL-1, a concentration of 15 ng/ml, which is sufficient to depress CYP1A activity, did not release detectable amounts of NO, but a concentration of 50 ng/ml resulted in the production of 15.8 ± 1.5 nmol of NO₂/mg of protein.

	Discussion

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The administration of LPS to both animals and humans is known to modulate the activity of a variety of cytochrome P450 enzymes in both peripheral organs and in the brain (Shedlofsky et al., 1994; Morgan, 1997; Renton et al., 1999; Renton and Nicholson, 2000). When added to isolated astrocytes, LPS has been shown to alter the activity of CYP1A1/2 and CYP2E1 isoforms (Tindberg et al., 1996; Nicholson and Renton, 1999). In this article, we used cultured astrocytes as a model system to investigate the role of cytokines in the LPS-evoked decrease in CYP1A activity in the brain. The results reported in this article demonstrate that CNS-derived cells respond directly to an immune stimulus (LPS) and this response probably involves the release and action of acute phase cytokines. A pathway involving such mediators is a major candidate to explain the decrease in CYP1A activity in the brain that is observed during localized inflammation (Nicholson and Renton, 2001).

The effects of LPS on hepatic cytochrome P450 activity have been well established and characterized. Several lines of research have convincingly demonstrated that these effects result from the stimulation of immune responses in the periphery. Ghezzi et al. (1986) performed serum transfer experiments in a strain of LPS-resistant mice to demonstrate that LPS itself did not directly mediate the depression in cytochrome P450 but rather that a serum mediator, specifically IL-1, was the critical mediator. Paton and Renton (1998) demonstrated that hepatoma cells did not respond to LPS directly; however, when incubated with medium from macrophages stimulated by LPS, they responded by decreasing CYP1A activity. In comparison with the periphery, the brain has a much subtler immune response that is activated by insults, such as brain trauma and CNS infections (Matyszak, 1998). This immune response is characterized by the activation of astrocytes and microglia in a process termed gliosis (Andersson et al., 1992). This results in the generation of several intermediate products, such as acute phase cytokines, NO via the increased expression of inducible nitric oxide synthase, and stimulation of the arachidonic acid cascade (Gottschall et al., 1992; Matyszak, 1998; Lopez-Figueroa et al., 2000).

Many of the effects of LPS (peripherally and centrally) are known to occur through the generation of intermediate products, such as cytokines, proteases, free radicals, and prostaglandins (Kielian and Blecha, 1995; Montero-Menei et al., 1996; Paludan, 2000). It is likely that the effect of LPS on CYP1A activity in the model described here occurs through the stimulation of astrocytes and the subsequent generation these types of intermediate products.

The synthetic glucocorticoid DEX is a commonly used anti-inflammatory drug that prevents cytokine synthesis by activating the transcriptional inhibitor I-B (Scheinman et al., 1995). When primary cultures of astrocytes were incubated with DEX concurrently with LPS, there was a complete prevention of the LPS-induced decrease in CYP1A activity. In addition, DEX dramatically decreased the release of two major acute phase cytokines, TNF- and IL-1, normally released after incubation with LPS. These observations support the idea that acute phase cytokines could mediate the effects of LPS on CYP1A activity in astrocytes. Incubation of cultured astrocytes with TNF- or IL-1 in a concentration similar to that produced by cells in response to LPS could partially mimic the effects of LPS on CYP1A activity and, thus, may contribute to this response. This also supports the idea that LPS decreases CYP1A activity in astrocytes via cytokine production. IFN- dramatically decreased CYP1A activity in cultured astrocytes, implicating it as a potential mediator of these effects. However, IFN- could not be detected in medium from cultured cells stimulated by LPS. This result was surprising because IFN- is released in vivo after LPS administration (Ho, 1964; Nicholson and Renton, 2001). It seems that cultured astrocytes, although very sensitive to this cytokine, do not release it in response to LPS. Thus, IFN- is unlikely to contribute to the effects of LPS on CYP1A activity in cultured astrocytes.

In contrast, no effect of IL-6 or IL-1 was observed on CYP1A activity. The effects of IL-6 on cytochrome P450 activity have been widely reported but are contrasting in nature. Earlier work using in vitro models has shown IL-6 to be capable of depressing the activity of a variety of cytochrome P450 isoforms (Williams, 1991; Fukuda et al., 1992; Fukuda and Sassa, 1994). However, the effects of IL-6 become less clear when administered in vivo because it has a variety of different effects depending on the isoform examined (Morgan, 1991; Morgan et al., 1994). IL-6, at the concentrations examined in this article, did not affect CYP1A activity in isolated astrocytes, indicating that this cytokine does not seem to mediate the effects of LPS in this model. It is also surprising that IL-1 had no effect on CYP1A activity in astrocytes because the biological activities of both IL-1 isoforms are mediated through the same receptor and IL-1-depressed CYP1A activity (Anforth et al., 1998). Earlier work in vivo also showed an inability of IL-1 to modulate CYP1A activity in brain whole-membrane fractions (Nicholson and Renton, 2001). In addition, it has been reported that the relative potencies of these two isoforms of IL-1 are not necessarily identical (Anforth et al., 1998). Of the major acute phase cytokines released during inflammation, it has become apparent that at least two of these cytokines, IL-1 and TNF-, can be implicated in the LPS-evoked decrease of CYP1A in cultured astrocytes.

Astrocytes respond to an immune challenge by the synthesis and release of immune mediators, such as the acute phase cytokines (Sawada et al., 1989; Chung and Benveniste, 1990). These cytokines in turn can act in an autocrine or paracrine manner to affect a decrease in CYP1A activity. In the cellular make-up of the glial cultures used here, the vast majority of cells (95%) are astrocytes, with a minor contribution of microglia (3%) and oligodendrocytes (2%). It is possible that cytokines, produced by activated microglia, may participate in the loss of CYP1A activity in astrocytes. The contribution of cytokines released from this cell-type, however, is likely to be relatively minor because the percentage of microglia in this culture system is fairly small.

Another potential mediator that may be involved in the loss of CYP1A by LPS is NO, which is released by LPS in these cultures. It has been well established that substantial quantities of NO are released during inflammation and in response to immune stimuli, such as LPS (Boje and Arora, 1992; Chao et al., 1992; Zielasek et al., 1992). It has been reported that cytokines induce the release of NO from immunocompetent cells, and thus, it is possible that NO, released by astrocytes, is responsible for part or all of the effects of cytokines on CYP1A activity. However, the levels of NO produced in astrocytes treated with cytokines were very low or negligible. Measurable amounts of NO are only detectable after treatment of cultures with 50 ng/ml IL-1, whereas suppression in CYP1A activity in these cultures occurred at a much lower dose (15 ng/ml), which produced undetectable levels of NO. These observations suggest that the actions of acute phase cytokines on CYP1A activity in astrocyte cultures do not involve an NO-dependent mechanism.

These results implicate cytokines as playing a major role in the LPS-induced decrease in CYP1A activity in cultured astrocytes. Because neither TNF- nor IL-1 could fully mimic the effects of LPS on CYP1A activity, it is likely that other mediators might work in consort with them to cause this down-regulation in activity.