Abstract
The effects of oncostatin M on the expression of different cytochrome P450 (CYP) isozymes has been investigated in human hepatocytes. The dose-response and time-course analyses of effects on CYP1A2 and CYP3A4 isozymes revealed that maximal inhibition was reached after 48 hr of exposure of human hepatocytes to 25 units/ml oncostatin M. Reductions in CYP1A2 and CYP3A4 activity produced by oncostatin M correlated with decreases in protein content, de novo protein synthesis and specific mRNA levels, thus suggesting that oncostatin M could down-regulate CYP expression at the transcriptional level. The inhibitory potency of oncostatin M on CYP expression was compared with that of other cytokines belonging to the interleukin-6 receptor family (interleukin-6, interleukin-11 and leukemia inhibitory factor), and interferon-γ, which is recognized to inhibit human CYP expression, and granulocyte colony-stimulating factor, a cytokine that shares structural homology with the interleukin-6 family but has a different transduction signal. Maximal reductions in CYP1A2 activity were reached after 48 hr of treatment with cytokines. At that time, oncostatin M showed the highest inhibitory effects on CYP1A2 activity (38% of control), followed by interferon (49% of control) and interleukin-6 (60% of control), whereas minor effects were produced by the other cytokines (74–80%). Comparable decreases were observed for CYP2A6, CYP2B6 and CYP3A4 activities. Enzymatic activity and de novoprotein synthesis of 3-methylcholanthrene-induced CYP1A2 and dexamethasone-induced CYP3A4 were also reduced to a much greater extent by oncostatin M than by other cytokines. The results show that oncostatin M is the most effective cytokine in down-regulating CYP isozymes in human hepatocytes, and its effects were evident even after removal of the cytokine from the culture medium.
Profound systemic and hepatic functional changes are derived from the inflammatory response of a host to insults of traumatic, infectious or immune origin (Kushner, 1982). This response is characterized in the liver by increases in synthesis and secretion of acute-phase proteins, alterations of intermediate metabolism and inhibition of xenobiotic biotransformation (Koj, 1985; Kushner and Mackiewicz, 1987;Richards and Gauldie, 1994). The decrease in the rate of drug metabolism during the inflammatory reaction arises from the reduced activity of specific CYP isoenzymes (Morgan, 1993; Shedlofsky et al., 1994). These effects are reproduced after treatment of laboratory animals with immunoactivators, including cytokines and cytokine-releasing agents (Ghezzi et al., 1985; Craiget al., 1990; Ansher et al., 1992; Chenet al., 1992). Despite the widely accepted belief that cytokines can inhibit the CYP system in vivo, the mechanism of this effect is largely unknown and could be mediated or influenced by other cytokines or factors either released or coadministered during the inflammation process.
In vitro studies with hepatic cells, and in particular human hepatocyte cultures, offer defined systems for studying the direct effects of individual cytokines on the regulation of hepatic CYP expression and activity in man. IL-6 (Williams et al., 1991;Abdel-Razzak et al., 1993; Fukuda and Sassa, 1994; Clarket al., 1995), IL-1β (Barker et al., 1992;Abdel-Razzak et al., 1993; Clark et al., 1995;Muntané-Relat et al., 1995) and TNF-α (Abdel-Razzaket al., 1993; Chen et al., 1995; Muntane-Relatet al., 1995), the three major proinflammatory cytokines, down-regulate CYP expression in rodent and human hepatic cells in culture. It has also been shown that other cytokines, including IFN (Donato et al., 1993a, 1997; Abdel-Razzak et al., 1993, Clark et al., 1995) and TGF-β (Abdel-Razzak et al., 1994), can influence CYP isozymes in cultured hepatocytes.
OSM is a multifunctional cytokine that is structurally and functionally related to IL-6 (Rose and Bruce, 1991; Hibi et al., 1996). The overlapping biological effects of OSM and IL-6 in many cellular systems have been explained by their sharing the same signal transducing molecule gp130 (Gearing et al., 1992; Liuet al., 1992; Kishimoto, et al., 1994). OSM acts on a wide variety of cells and elicits diversified biological responses such as growth regulation of certain tumor and non-tumor-derived cell lines (Zarling et al., 1986; Horn et al., 1990), induction of differentiation of several cell types (Rose and Bruce, 1991; Brown et al., 1991), low-density lipoprotein up-regulation in liver cells (Grove et al., 1991) and acute-phase protein induction in hepatocytes (Richards et al., 1992). However, despite the available information on the multiple hepatocellular functions of OSM, its effects on hepatic drug metabolism remain unknown.
The present study was conducted to examine the potential down-regulation by OSM of CYP expression. To this end, specific monooxygenase activities for different CYP isozymes, de novoCYP protein synthesis and specific mRNA expression were examined. The effects of OSM have also been compared with those of IL-6 and IFN, two cytokines with well-known depressing effect on CYP expression, as well as with the other IL-6-related cytokines, IL-11 and LIF (Nicola, 1994;Kishimoto et al., 1994; Hibi et al., 1996), and with G-CSF, a cytokine that shares structural homology with the IL-6 family but has a different transduction signal (Rose and Bruce, 1994). The results indicate that OSM has very potent inhibiting effect on human CYP expression in basal and induced hepatocytes and is even more effective than other cytokines with demonstrated capacity to down-regulate CYP proteins.
Materials and Methods
Materials.
Recombinant human OSM was obtained from Pharmigen (San Diego, CA); recombinant human IL-6 was purchased from Menarini Diagnostics (Florence, Italy); recombinant human IL-11, LIF and G-CSF were from British Bio-technology Products (Oxon, UK); recombinant human IFN, 7-benzoxyresorufin, 7-ethoxyresorufin, collagenase and β-glucuronidase/arylsulfatase were purchased from Boehringer-Mannheim (Mannheim, Germany); coumarin, 7-hydroxycoumarin, resorufin, testosterone and MC were purchased from Sigma Chemical (St. Louis, MO); DEX was obtained from Merck Sharp & Dohme (Alcalá de Henares, Madrid); 6β-hydroxytestosterone was supplied by Steraloids (Wilton, NH); trans-35S-label (specific activity, 1138 Ci/mmol) was obtained from ICN Pharmaceuticals (Costa Mesa, CA); newborn calf serum was obtained from GIBCO (Paisley, UK); Ham’s F-12 and Leibovitz L-15 culture media were from Flow (Irvine, UK); RPMI 1640 methionine-free medium was purchased from Seromed (Berlin, Germany); and all other reagents used in this study were of analytical grade.
Isolation and culture of human hepatocytes.
Surgical liver biopsies (1–5 g) were taken from patients undergoing cholecystectomy after informed consent was obtained. Patients had no known liver pathology, nor did they receive medication during the weeks before surgery. None of the patients were habitual consumers of alcohol or other drugs. A total of 11 liver biopsy samples (from four men and seven women) were used. Patients’ ages ranged from 33 to 70 years (table 1). Human hepatocytes were isolated using a two-step perfusion technique (Gómez-Lechónet al., 1990) and seeded onto 24-well or 3.5-cm-diameter fibronectin-coated plates (3.6 μg/cm2) at a density of 8 × 104 cells/cm2 in an appropriate volume of medium. Culture medium was Ham’s F-12/Leibovitz L-15 (1/1, v/v) supplemented with 2% newborn calf serum, 5 mM glucose, 50 units/ml penicillin, 50 μg/ml streptomycin, 0.2% bovine serum albumin and 10−8 M insulin. Medium was changed 1 hr later to remove unattached hepatocytes. By 24 hr, cultures were shifted to serum-free medium.
Characteristics of donors and of liver cell preparations
Treatment of cultures.
Cytokines were prepared as sterile solutions in culture medium containing serum and added directly to cultures. Treatments were started by 24 hr of culture after medium renewal, and control cells were run in parallel. For CYP induction experiments, MC and DEX were dissolved in dimethylsulfoxide and added to 24-hr-old cultured human hepatocytes at a final concentration of 2 and 1 μM, respectively (concentration of solvent in culture medium was 0.5% v/v).
Monooxygenase activity assays.
CYP1A2 and CYP2B2 activities were assessed as 7-ethoxyresorufin-O-deethylation (Gonzalez, 1990) and 7-benzoxyresorufin-O-debenzylation (Waxman et al., 1991), respectively. Activities were assayed in intact hepatocytes cultured on 24-well culture plates incubated with 8 μM 7-ethoxyresorufin or 15 μM 7-benzoxyresorufin, respectively, and the resorufin formed was quantified fluorimetrically as previously described (Donato et al., 1993b). CYP2A6 activity (assessed as coumarin 7-hydroxylation, Yun et al., 1991) was measured directly in intact hepatocytes cultured on 24-well culture plates incubated with 100 μM coumarin for 30 min at 37°C, and the 7-hydroxycoumarin formed was quantified fluorometrically as described (Donato et al., 1997). CYP3A4 activity (assessed as testosterone 6β-hydroxylation; Waxman et al., 1991) was measured by incubating intact hepatocytes cultured on 24-well plates for 30 min with 300 μl of culture medium containing 250 μM testosterone. Metabolites were extracted and analysed by HPLC as described (Donatoet al., 1993a). Cellular protein was measured according to the method of Lowry et al. (1951).
Analysis of CYP proteins.
Polyclonal antibodies against recombinant CYP1A2 and CYP3A4 were kindly provided by Dr. F. P. Guengerich (Nashville, TN). For Western blot analysis, cellular lysates were obtained from human cultured hepatocytes incubated with cytokines for the indicated times and electrophoresed in a SDS-polyacrylamide gel (30 μg of protein/lane). Proteins were transferred to Immobilon membranes (Millipore), and sheets were sequentially incubated with goat antiserum raised against recombinant CYP1A2, rabbit antiserum raised against goat-IgG and with horseradish peroxidase-labeled rabbit-IgG, or with rabbit antiserum raised against recombinant CYP3A4 and horseradish peroxidase-labeled rabbit-IgG. The substrate for the peroxidase enzyme was 0.05% diaminobenzidine (w/v) and 0.001% H2O2 (v/v) in PBS. The relative intensities of the bands were estimated from densitometric analysis of the blot with an Image Analyzer (Visilog 4; Noesis Vision, Velizy, France).
For immunoprecipitation assays, hepatocytes stimulated with appropriate concentrations of cytokines for the indicated times were shifted to methionine-free RPMI-1640 medium and pulse-labeled for 4 hr with 50 μCi/ml of [35S]-methionine (trans35S-label). Radiolabeled CYP isozymes present in the cellular lysate were immunoprecipitated with specific polyclonal antibodies against recombinant CYP1A2 and CYP3A4, subjected to SDS-polyacrylamide electrophoresis under reducing conditions and fluorography as described (Castell et al., 1990). The radioactive bands were excised and quantified.
Analysis of mRNA by semiquantitative RT-PCR.
Total cellular RNA was extracted and reverse transcribed as described (Chomczynski and Sacchi, 1987). For CYP3A4 cDNA amplification (Gonzalezet al., 1988), the forward primer was from 1353 to 1379 nt (5′-CCT TAC ACA TAC ACA CCC TTT GGA AGT-3′), and the reverse primer was from 1705 to 1734 nt (5′-AGC TCA ATG CAT GTA CAG AAT CCC CGG TTA-3′) and amplified a predicted 382-bp fragment. For CYP1A2 cDNA (Jaiswalet al., 1986), the forward and reverse primers 5′-AAC AAG GGA CAC AAC GCT GAA T-3′ and 5′-GGA AGA GAA ACA AGG GCT GAG T-3′, respectively, produced a predicted 453-bp fragment between position 1178 and 1630. In parallel, we analyzed G6PD (Persico et al., 1986) as an internal control for normalization. The forward and reverse primers, 5′-AAG CCC GCC TCC ACC AAC TCA-3′ and 5′-GGC ACC CCA TCC CAC CTC TCA T-3′, respectively, produced a predicted 236-bp fragment between position 1293 and 1529. Diluted cDNA (3 μl) was amplified in 30 μl of 10 mM Tris-HCl (pH 8.8) containing 50 mM KCl, 1.5 mM MgCl2, 0.1% Triton X-100, 60 μM concentration of each deoxynucleotide triphosphate, 1 unit of DNA polymerase (Dynazyme II, Finnzymes OY) and 0.2 μM concentration of each primer. Amplification was performed in a Peltier Thermal Cycler (PTC-100HB, MJ Research) programmed for an initial denaturation of 4 min at 94°C, followed by 27 cycles of 45 sec at 94°C, 45 sec at 58°C and 1 min at 72°C and a final extension of 5 min at 72°C. Appropriate dilutions were empirically determined for each cDNA to ensure that the resulting PCR product was derived only from the exponential phase of the amplification. For quantitative analysis, aliquots of the PCR reaction were subjected to electrophoresis on agarose gel, and the products were visualized by ethidium bromide staining. The gel image was analysed and quantified with the VisiLog Software Package (VISILOG 4).
Determination of nitrite concentration.
To determine the amount of nitric oxide synthetized by hepatocytes, the culture supernatants were assayed for nitrite, as a stable end product of nitric oxide oxidation. Nitrite accumulation was measured in 96-well plates by adding 100 μl of culture supernatant to 100 μl of Griess reagent as described previously (Donato et al., 1997).
Statistical analysis.
Each experiment was done in at least three cell preparations from different donors. Data are shown as the mean ± S.D. Data were analyzed by using the Student’st test. Values of P < 0.05 were considered significant.
Results
Down-regulation of CYP1A2 and CYP3A4 expression in human hepatocytes by OSM.
The CYP1A2 and CYP3A4 activities of freshly isolated hepatocytes (2.49 ± 0.62 and 136 ± 33 pmol/mg/min, respectively; n = 6) decreased during the first 24 hr in culture to 1.62 ± 0.46 and 95 ± 24 pmol/mg/min, respectively (n = 7). After this decrease, probably due to the adaptation of cells to culture conditions, the enzyme was stable up to 96 hr of culture. Incubation of human hepatocytes with increasing concentrations of OSM resulted in a dose- and time-dependent decrease in CYP1A2 and CYP3A4 activities. As seen in figure1, OSM had a maximal inhibitory effect on CYP1A2 (∼55% of control) and CYP3A4 activity (70% of control) in the 25 to 50 units/ml range after 48 hr of continuous exposure to the cytokine.
Dose-dependent and time-course effects of OSM on CYP1A2 and CYP3A4 activities. A, The 24 hr cultures were exposed to increasing concentrations of OSM, and enzymatic activities were measured 48 hr later. Results are expressed as a percentage of activity in cells maintained in control conditions for the same period of time (control values were 1.57 ± 0.37 and 89 ± 35 pmol/mg/min for CYP1A2 and CYP3A4, respectively). B, The 24 hr cultured hepatocytes were exposed to 25 units/ml OSM, and CYP1A2 and CYP3A4 activities were measured at the indicated times. Results are expressed as a percentage of activity in cells maintained in control conditions for the same period of time. Values represent mean ± S.D. of three cell preparations from different donors.
In parallel, the protein levels of CYP1A2 and CYP3A4 were examined in cellular lysates after electrophoretic separation of proteins, blotting on nylon membranes and immunodetection with specific polyclonal antibodies. As shown in figure 2A, OSM reduced the measurable levels of CYP1A2 and CYP3A4 in human hepatocytes. Experiments designed to measure the de novoprotein synthesis (fig. 2B) indicated that the synthesis of both CYP1A2 and CYP3A4 decreased as the concentration of OSM in the culture medium increased and reached a plateau by 48 hr (fig. 2C). In contrast, with results on CYP activities, protein synthesis of CYP1A2 is less affected than CYP3A4 synthesis by OSM.
Dose-dependent and time-course effects of OSM on CYP1A2 and CYP3A4 expression. After 24 hr in culture, hepatocytes were exposed to increasing concentrations of OSM for an additional 48 hr (A and B) or to 25 units/ml OSM for different times (C and D). A, Relative protein levels were estimated by Western blot analysis of cellular lysates with specific polyclonal antibodies and quantification of the blot by densitometric analysis. B and C, De novo protein synthesis was measured by specific immunoprecipitation of cellular lysates from cells previously labeled with 50 μCi of [35S]methionine for 4 hr in methionine-free RPMI-1640 culture medium. D, Changes in specific mRNAs were performed by semiquantitative RT-PCR. Values were calculated from confirmed three or four RT-PCR bands and are expressed as percentage of mRNA CYP/mRNA β-actin in control cells. Values are mean ± S.D. of three different experiments and are expressed as a percentage of respective control values.
To better understand the down-regulation of CYP expression by OSM, a series of experiments were conducted in human hepatocytes to determine whether this event could occur at the pretranscriptional level. Analysis of specific mRNAs by semiquantitative RT-PCR revealed a significant reduction in CYP1A2 and CYP3A4 mRNAs at 6, 24 and 48 hr after incorporation of OSM to the hepatocyte culture medium (fig. 2D). Taken together, the results support the idea that the decrease in the abovementioned enzyme activities reflects a lower concentration of both CYPs, presumably as a consequence of a reduced expression of specific CYP mRNAs and protein synthesis.
Comparative effects of cytokines on the activity of different CYP enzymes in human hepatocytes.
The ability of other cytokines to modulate CYP1A2 activity in primary cultures of human hepatocytes was compared with that of OSM. After 24 hr in culture, cells were incubated with increasing concentrations of OSM, IFN, IL-6, LIF, IL-11 and G-CSF, and CYP1A2 activity was measured 24 hr later. As seen in figure3A, 50 units/ml OSM decreased CYP1A2 activity to 69% of control, whereas at same concentration the other cytokines caused a lower inhibition (83–97% of control). On the basis of the effects of the cytokines on CYP1A2 activity, 25 units/ml OSM, 100 units/ml IL-6, 100 units/ml LIF, 75 units/ml IL-11, 300 units/ml IFN and 320 units/ml G-CSF were selected for further experiments.
Dose-response and time-course effects of cytokines on the CYP1A2 activity. A, The 24 hr cultured hepatocytes were exposed to increasing concentrations of cytokines and CYP1A2 activity was measured 24 hr later. Results are expressed as a percentage of activity in cells maintained in control conditions (1.49 ± 0.50 pmol/mg/min) for the same period of time. B, The 24 hr cultured hepatocytes were exposed to 25 units/ml OSM, 100 units/ml IL-6, 100 units/ml LIF, 75 units/ml IL-11, 300 units/ml IFN or 320 ng/ml G-CSF and CYP1A2 activity was measured at the indicated times. Results are expressed as a percentage of respective controls activity in cells maintained in control conditions by the same period of time. Values are the mean of three different experiments.
A time-course study revealed that maximal effects on CYP1A2 activity were achieved after 48 to 72 hr of exposure of human hepatocytes to cytokines (fig. 3B). At 48 hr, the greatest decrease in CYP1A2 activity was obtained with OSM (38% of control activity), followed by IFN (49%) and IL-6 (60%), whereas minor effects were produced by LIF (74%), IL-11 (76%) and G-CSF (80%). These effects on CYP1A2 activity were paralleled by changes in de novo synthesis of CYP1A2 (data not shown).
The inhibitory effect of OSM on CYP1A2 and CYP3A4 (fig. 1) was also investigated on CYP2A6 and CYP2B6 and compared with that of other cytokines. As shown in table 2, OSM and IFN caused strong reductions in all the CYP activities (ranging from 38% to 70% and from 48% to 66% of control activities, respectively). Exposure of hepatocytes to IL-6 caused marked decreases only in CYP1A2 and CYP2B6 activities, whereas the effects on the other activities were minor. Treatment with IL-11, LIF or G-CSF produced no or very reduced changes in CYP activities.
CYP-dependent monooxygenase activities of human hepatocytes treated with cytokines
Effects of cytokines on the inducible expression of CYP isozymes in human hepatocytes.
To analyze the effects of cytokines on MC-inducible CYP1A2 and DEX-inducible CYP3A4, 24-hr human hepatocytes were incubated with inducers and cytokines for an additional 48 hr. CYP1A2 activity increased after MC treatment (6-fold over noninduced controls). This induction was partially prevented when cytokines were present in the incubation media (fig.4A). OSM and IFN were the most effective cytokines in preventing CYP1A2 induction by MC (43% and 68% of MC-treated hepatocytes respectively), whereas IL-11, LIF and G-CSF had no effects. De novo synthesis of CYP1A2 was studied in the same cultures. MC-treated cells increased the synthesis of CYP1A2. However, the synthesis of the enzyme dropped to the levels of uninduced cells in cultures where OSM or IFN was present (fig. 4B).
Effects of cytokines on activity and protein synthesis of MC-induced CYP1A2. After 24 hr in culture, human hepatocytes were shifted to culture medium containing 2 μM MC and exposed to cytokines (for details, see legend to fig. 3). CYP1A2 activity (A) and de novo synthesis of CYP1A2 (B) were measured 48 hr later as described in the text. Results are expressed as a percentage of cells maintained in control conditions for the same period of time. Values are the mean ± S.D. of three experiments.
The CYP3A4 activity increased 2-fold in hepatocytes incubated with DEX compared with noninduced cells (fig. 5A). Cytokines also antagonized this induction, with OSM and IFN showing the strongest inhibitory effect (47% and 80%, respectively, of DEX-treated cell values). Changes in enzyme activity were paralleled by changes in CYP3A4 synthesis. The presence of OSM in the culture medium reduced the de novo synthesis of CYP3A4 to 56% of DEX-treated cells (fig. 5B). IFN, IL-6 and LIF had a moderate inhibitory effect (76–84% of control value), whereas IL-11 and G-CSF had no significant effects on DEX-induced CYP3A4 synthesis.
Effects of cytokines on activity and protein synthesis of DEX-induced CYP3A4. After 24 hr in culture, human hepatocytes were shifted to culture medium containing 1 μM DEX and exposed to cytokines (for details, see legend fig. 3). CYP3A4 activity (A) and de novo synthesis of CYP3A4 (B) were measured 48 hr later as described in the text. Result are expressed as a percentage of cells maintained in control conditions for the same period of time. Values are the mean ± S.D. of three experiments.
Maintenance of effects produced by OSM on CYP1A2 activity and protein synthesis.
The maintenance of the signal produced by OSM was studied in two parallel sets of experiments in which kinetics of CYP1A2 activity and de novo synthesis were evaluated. After 24 hr of treatment with 25 units/ml OSM, human hepatocytes were either incubated in presence of the cytokine or shifted to control culture conditions for an additional period of time. The decrease in both CYP1A2 activity and protein synthesis produced by OSM was maintained for at least 72 hr after removing the cytokine from the culture medium (fig. 6). The time course of this effect was similar to that of the activity and protein synthesis reductions observed in cells continuously exposed to OSM.
Maintenance of effects produced by OSM on activity and protein synthesis of CYP1A2. After 24 hr in culture, hepatocytes were treated with 25 units/ml OSM for additional 24 hr. Cells were then shifted to control culture medium or maintained in presence of 25 units/ml OSM. At the indicated times, CYP1A2 activity (A) and de novo synthesis (B) were measured as described in the text. Results are expressed as a percentage of activity and protein synthesis at 24 hr of culture in control cells. Data represent the mean ± S.D. of three different cultures.
Nitrite levels after hepatocytes exposure to cytokines.
The possible role of induced nitric oxide biosynthesis in cytokines effects on the CYP system was examined. After nitric oxide production by hepatocytes, the accumulation of nitrite released into culture medium was measured. The results given in table3 show that after 24 hr of treatment with cytokines, only IFN led to the induction of nitric oxide synthesis. None of the other cytokines produced alterations of nitrite levels in culture supernatant even after longer exposure periods (data not shown).
Effect of cytokines on nitric oxide synthesis by human hepatocytes
Discussion
Most information on drug metabolism impairment during inflammation has been obtained in rodent in vivo or in vitromodels, and only a few studies have examined the effect of cytokines on human CYP expression (Abdel-Razzak et al., 1993, 1994;Donato et al., 1993a; Muntané-Relat et al., 1995). Most of these studies have focused on the effects of IFNs and the major inflammatory cytokines, namely, IL-6, IL-β and TNF-α. However, other cytokines, such as OSM, LIF, IL-11 and G-CSF, that are released during the acute-phase response have not yet been tested for their possible ability to influence the expression of human CYPs. In the present report, we provide the first evidence that OSM can both decrease basal CYP1A2 and CYP3A4 gene expression and strongly inhibit induction by MC or DEX, respectively. This cytokine was used at a concentration previously reported to mediate inhibition of albumin and increase positive acute-phase protein secretion (haptoglobin, α1-antichymotrypsin and fibrinogen) in HepG2 cells and rat hepatocytes (Richards et al., 1992). These findings complemented previous studies showing that IFN, TGF-β and inflammatory cytokines (IL-6, IL-1β and TNF-α) depressed CYP-associated drug metabolism in humans.
Although a general reduction in monooxygenase activity was derived from cytokine treatment of human hepatocytes, marked differences in the magnitude of the depression produced by each cytokine were found (fig.3, table 2). OSM proved to be the most effective cytokine in reducing all specific CYP activities. Its effects were even stronger than those produced by IFN and IL-6, two cytokines with known inhibitory action on human CYP gene expression in vitro (Donato et al., 1993a; Muntané-Relat et al., 1995). Parallel to activity inhibition, decreases in de novo synthesis and levels of CYP1A2 and CYP3A4 proteins also were observed (fig. 2). These results suggest that the alterations in monooxygenase activities observed after cytokine treatment could be due to decreases in enzyme content.
The expression of CYP proteins can be regulated transcriptionally or affected by processes altering the rate of protein stabilization and/or degradation. RT-PCR analysis showed that OSM treatment produced a down-regulation of the specific CYP1A2 and CYP3A4 mRNA that could entirely account for the loss of CYP protein and activity observed at the same time (fig. 2). The correlation of mRNA level, protein content and monooxygenase activity reported in this study supports the hypothesis that the down-regulation of CYP isozymes produced by OSM occurs predominantly at the pretranslational level. Whether OSM reduced CYP mRNA levels by interfering with the transcriptional activation of the genes or by increasing the rate of degradation of the mRNAs is not known. However, there is no reason to rule out the possibility that cytokines negatively regulate CYP genes by similar mechanisms to those involved in the induction of acute-phase genes. It is generally accepted that cytokines involved in inflammatory response appear to supress CYP gene expression largely by a transcriptional mechanism (Barker et al., 1992; Morgan et al., 1994), but other mechanisms may be involved. A decline in free heme content as a result of the induction of heme oxygenase, a catabolic enzyme of the heme group, seems to be an additional mechanism for the down-regulation of IL-6-mediated CYP gene expression (Fukuda and Sassa, 1994). Inhibition of CYP by IFN via an increase in xanthine oxidase activity and the generation of oxygen free radicals that subsequently destroy CYP also were proposed (Ghezzi et al., 1985). Recent studies have pointed out that the inhibitory effects produced by IFN and other inflammatory cytokines on CYP activities in rat (Stadleret al., 1994) and human (Donato et al., 1997) hepatocytes can be mediated by nitric oxide. This short-lived mediator is synthesized by hepatocytes during inflammation in response to cytokines (Billiar et al., 1990; Nussler et al., 1992). Our results showed that the only cytokine that significantly induced nitric oxide release by human hepatocytes is IFN (table 3), which suggests that the effects produced by OSM and the other cytokines studied on the CYP system are not mediated by nitric oxide biosynthesis.
Cytokines of the IL-6-related family that were studied in the present work, such as OSM, LIF and IL-11, exhibit multiple functions and redundancy in biological activities during the acute-phase reaction, immune response, hematopoiesis and so on (Liu et al., 1992;Rose and Bruce, 1994). On the basis of the results discussed above, we suggest that another of the common biological actions of this cytokine family is a general inhibition of expression of the CYPs. However, this effect is particularly relevant in the case of OSM. The mechanism by which cytokines negatively control CYP expression and other biological activities remains unknown, but it has been suggested that it is mediated by cytokine interactions with their respective plasmatic receptors (Kishimoto, 1994; Chen et al., 1995; Tinelet al., 1995). Stimulation by these cytokines of their specific receptors induces oligomerization and activation of receptor components. Specific IL-6 family receptors become associated with a common signal transducing receptor component, gp 130 (Kishimotoet al., 1994; Hibi et al., 1996). The gp 130 protein serves as a signal transducer not only for the IL-6 cytokine but also for LIF, OSM, IL-11 and ciliary neurotrophic factor, which suggests that it is the cause of the common biological functions that these cytokines carry out in various tissues.
It is important to identify the mechanisms that mediate the decreases in the capacity of the liver to metabolize drugs. The pharmacological and toxicological implications of the profound alterations of constitutive and inducible forms of CYPs produced by cytokines are evident because the action on CYP functionality could impair the elimination of drugs subsequently administered and alter their therapeutic efficacy. When OSM effects on human hepatocytes are considered in the context of events that occur during inflammation, it seems conceivable that OSM could play an important role in the control of the hepatic CYP system during the inflammatory process. This potential in vivo down-regulation of CYP expression by OSM could be mediated by two different mechanisms. First, by direct interaction with its specific receptor (Mosley et al., 1996), activation of specific receptors of other related cytokines (LIF; Gearing et al., 1992) or regulation of the mRNA of the IL-6 receptor (Geisterfer et al., 1995). Second, it could also exert a cumulative indirect action on the CYP system in vivo by increasing circulating levels of IL-6 viaaction on vascular endothelial cells or fibroblasts (Brown et al., 1991; Richards and Agro, 1994) for a wide variety of cell types, including endothelial and fibroblasts cells that have a specific receptor (Richards and Agro, 1994). In addition, the biological signal produced in vivo by OSM might be maintained in time for our results show that OSM inhibition of CYP1A2 activity and protein synthesis was maintained for at least 72 hr after incubation with OSM ended (fig. 7). This effect contrasts with our previous studies indicating that the effects of IFN, a cytokine with a receptor that is not the same as the of the IL-6 receptor family, on CYP1A2 activity are transient, and enzyme activity was fully restored 24 hr after IFN elimination from culture medium (Donato et al., 1993a). The potent activity shown by OSM in the regulation of the human CYP system provides us with a good reason for doing additional research into drug interactions and human liver functionality.
Acknowledgments
The authors express their thanks to Dr. F. P. Guengerich (Center in Molecular Toxicology, Vanderbilt University, Nashville, TN) for providing anti-CYP1A2 and anti-CYP3A4 antibodies. The authors thank Dr. E. Offord for critical reading of this manuscript. The expert technical assistance of M. C. Lorenzo, T. Hualde and E. Belechón also is appreciated.
Footnotes
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Send reprint requests to: Dr. M. JoséGómez-Lechón, Unidad de Hepatologı́a Experimental, Centro de Investigación, Hospital Universitario La Fe, Avda. Campanar 21, Valencia-46009, Spain.
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↵1 The financial support of European Union (Project AIR2-CT93-0860, BMH1-CT94-1097 and BIO4-CT96-0052) and the ALIVE Foundation is gratefully acknowledged.
- Abbreviations:
- CYP
- cytochrome P450
- DEX
- dexamethasone
- G-CSF
- granulocyte colony-stimulating factor
- IFN
- interferon-γ
- IL-1β
- interleukin-1β
- IL-6
- interleukin-6
- IL-11
- interleukin-11
- LIF
- leukemia inhibitory factor
- MC
- 3-methylcholanthrene
- OSM
- oncostatin M
- RT
- reverse transcriptase
- PCR
- polymerase chain reaction
- TGF-β
- transforming growth factor-β
- TNF-α
- tumor necrosis factor-α
- Received June 30, 1997.
- Accepted December 15, 1997.
- The American Society for Pharmacology and Experimental Therapeutics
