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Research ArticleArticle

Influence of Extracellular Matrix Overlay and Medium Formulation on the Induction of Cytochrome P-450 2B Enzymes in Primary Cultures of Rat Hepatocytes

Edward LeCluyse, Peter Bullock, Ajay Madan, Kathy Carroll and Andrew Parkinson
Drug Metabolism and Disposition August 1999, 27 (8) 909-915;

Abstract

The effect of medium formulation, composition of extracellular matrix overlay, and culture dish material on liver microsomal cytochrome P-450 (CYP) 2B induction by phenobarbital (PB) was investigated in primary cultures of rat hepatocytes. When hepatocytes were maintained on Permanox dishes with an overlay of either collagen (type I) or Matrigel, Williams’ E medium was superior to other medium formulations in terms of the magnitude of induction of CYP2B on a per milligram microsomal protein basis. Modified Chee’s medium (MCM) and hepatocyte culture medium were intermediate in their capacity to sustain induction of CYP2B by PB, and Dulbecco’s modified Eagle’s medium was slightly less effective. The overall induction of CYP2B activity by PB was, on average, 50% lower in hepatocytes cultured on polystyrene dishes (LUX). Little or no difference was observed between hepatocytes overlaid with collagen and those overlaid with Matrigel. MCM was superior to Williams’ E medium in terms of the yield of microsomal protein and the ultrastructural features of the hepatocyte monolayers. CYP2B induction by PB was optimal after 3 days of treatment in either medium. CYP1A, CYP3A, and CYP4A activities could be induced in vitro by prototypical inducing agents in hepatocytes cultured on Permanox dishes with MCM and a Matrigel overlay to comparable levels observed in vivo. The results of these studies show that medium formulation and culture vessel material, but not the type of extracellular matrix overlay, have significant effects on the induction of CYP enzymes in cultured rat hepatocytes maintained in a sandwich configuration.

Over the past two decades significant efforts have been made to establish primary cultures of rat hepatocytes in which microsomal cytochrome P-450 (CYP)3 enzymes belonging to the CYP2B subfamily are maintained and/or induced as they are in vivo (LeCluyse et al., 1996b). In vivo liver microsomes from untreated rats contain undetectable levels of immunoreactive CYP2B1 and low levels (10–40 pmol/mg) of CYP2B2 (Thomas et al., 1987; Ryan and Levin, 1990). The levels of CYP2B1 and CYP2B2 in rat liver microsomes are coordinately regulated in a dose-dependent manner by phenobarbital (PB) and PB-type inducers (Adesnik et al., 1981; Waxman et al., 1990). The marked induction (∼50-fold) of CYP2B1 and CYP2B2 enzymes involves transcriptional activation of their respective genes by mechanisms that have yet to be fully elucidated.

Three factors have been consistently shown to have significant effects on CYP enzyme expression and induction in vitro. These are: 1) the biophysical nature and composition of the extracellular matrix (Ben-Ze’ev et al., 1988; Lindblad et al., 1991), 2) the formulation of culture medium (Jauregui et al., 1986; Turner and Pitot, 1989), and 3) the combination and concentration of hormones (Dich et al., 1988; Dahn et al., 1993). Unfortunately, direct comparisons of rat hepatocyte culture protocols designed to study CYP expression are complicated by the number of culture variables and the interdependence of substratum, extracellular matrix overlay, medium formulation, and hormone supplementation. Comparisons between various studies are also complicated by the endpoints used to monitor CYP expression. For example, Sidhu et al. (1993) have shown that modified Williams’ E medium (WEM) and modified Chee’s medium (MCM) are equally effective in supporting induction of CYP2B mRNA by PB, but the latter is superior in supporting induction of CYP2B protein and activity. The age of the cultures before inducers are introduced, the relative potency of inducers within each class, the final concentration of inducers in culture medium, and the duration of exposure to inducers also have profound effects on the induction of CYP enzymes in cultured hepatocytes. Therefore, the purpose of this study was to determine the effects of a number of culture conditions on the overall cell morphology and the induction of CYP2B enzymes by PB in long-term cultures of rat hepatocytes.

Four medium formulations, namely, Dulbecco’s modified Eagle’s medium (DMEM), WEM, MCM, and hepatocyte culture medium (HCM), were chosen to represent both conventional formulations, which are easily obtained commercially (WEM, DMEM) and “enriched” formulations, which contain superphysiological concentrations of certain components, such as amino acids (MCM, HCM). Collagen and Matrigel overlay were compared because both represent matrix components found in the space of Disse, yet are expressed during different stages of hepatocyte differentiation (laminin = fetal; collagen = mature; Reid et al., 1992). However, Matrigel is thought to be superior to collagen in its ability to restore the differentiated phenotype to cultured hepatocytes, presumably because of its complex nature (laminin, collagen type IV, entactin, etc.; Berry et al., 1991). Finally, two commonly used, commercially available types of culture dishes [polystyrene (LUX) and Permanox] were compared for their influence on PB-induced CYP2B enzyme expression.

Experimental Procedures

Materials.

All culture media, sera, glutaMAX media supplement, and nonessential amino acids were obtained from Gibco BRL (Grand Island, NY). Insulin, transferrin, selenium, linoleic acid, and BSA supplement (ITS+) and Matrigel were purchased from Collaborative Biomedical Research (Bedford, MA). Collagenase (CLS 2) was obtained from Worthington Biochemical Corporation (Lakewood, NJ). Collagen, type I (Vitrogen) was obtained from CelTrix (Santa Clara, CA). Petri dishes (60 mm) (LUX, Permanox) were purchased from NUNC (Naperville, IL). The sources of the various steroids and other reagents used in this study have been reported previously (Sonderfan et al., 1987; Pearce et al., 1996).

Isolation and Culture of Rat Hepatocytes.

Hepatocytes were isolated from rat liver by a modification of the collagenase digestion method described by LeCluyse et al. (1996a), which was adapted from the method of Quistorff et al. (1989). Briefly, rat livers were perfused with calcium-free buffer containing 5.5 mM glucose and 0.5 mM EGTA for 10 min at a flow rate of 35 to 40 ml/min followed by perfusion with buffer containing 1.5 mM calcium and collagenase (0.3–0.4 mg/ml) for 10 to 15 min at a flow rate of 30 to 35 ml/min. Hepatocytes were dispersed from the digested liver in DMEM containing 5% fetal calf serum, insulin (4 μg/ml), and dexamethasone (1 μM) and washed by low-speed centrifugation (50g, 2 min). Cell pellets from rat liver were resuspended in equal volumes of medium and 90% isotonic Percoll and centrifuged at 70g for 5 min. The resultant pellets were resuspended in fresh medium and washed once by low-speed centrifugation. Hepatocytes were resuspended in supplemented DMEM and viability was determined by trypan blue exclusion. Cell preparations were not used for experimental studies unless the viability was ≥85%.

Unless otherwise specified, approximately 3 × 106 hepatocytes were added to 60-mm culture dishes in 3 ml of serum-free medium containing 0.1 μM dexamethasone, 6.25 μg/ml insulin, 6.25 μg/ml transferrin, and 6.25 ng/ml selenium and allowed to attach for 2 to 3 h at 37°C in a humidified chamber with 95%/5% air/CO2. Culture dishes were gently swirled and medium containing unattached cells and debris was aspirated. Cultures were overlaid with either collagen or Matrigel as described elsewhere (LeCluyse et al., 1996a). Cultures were then returned to the humidified chamber. Medium was changed on a daily basis thereafter. In most cases, primary cultures of rat hepatocytes were maintained for 2 to 3 days before initiating treatment with PB.

Induction Studies.

Medium and overlay effects

Hepatocytes were plated onto Permanox or LUX culture dishes precoated with collagen, type I, according to the methods of Waxman et al. (1990). After cell attachment, monolayers were overlaid with either ice-cold neutralized collagen or ice-cold medium containing 0.25 mg/ml Matrigel as described by LeCluyse et al. (1996a). In addition, groups of hepatocyte cultures (n = 5–6 per treatment group) were maintained in one of four medium formulations: DMEM, WEM, MCM, or HCM. These procedures produced eight different groups of hepatocyte cultures based on type of medium and composition of the matrix overlay. Monolayers from each group were subsequently maintained for 2 to 3 days in the corresponding serum-free medium supplemented with ITS+ and 0.1 μM dexamethasone before initiating treatment with inducers. Groups of hepatocyte cultures were then treated for 3 consecutive days with either saline (control) or 100 μM PB. At the end of the study period, cells were harvested for the preparation of microsomes.

Time course of CYP enzyme induction.

Hepatocytes were plated onto Permanox culture dishes and overlaid with Matrigel as described above. Hepatocytes were maintained in MCM supplemented with ITS+ and 0.1 μM dexamethasone before initiating treatment with inducers. Groups of hepatocyte cultures (n = 5–6 dishes per treatment group) were then treated for 1 to 4 consecutive days with 0.1% dimethyl sulfoxide (control) or 100 μM PB. At the appropriate time point, cells from the corresponding groups were harvested for the preparation of microsomes.

Dose response of P-450 enzyme induction.

Hepatocytes were plated onto Permanox culture dishes and overlaid with Matrigel as described above. Hepatocytes were maintained in either WEM or MCM supplemented with ITS+ and 0.1 μM dexamethasone before initiating treatment with PB. Groups of hepatocyte cultures (n = 5–6 dishes per treatment group) were then treated for 3 consecutive days with either 10, 25, 100, 250, 750, or 1500 μM PB. Control cultures were treated with saline vehicle alone. At the end of the study period, cells were harvested for the preparation of microsomes.

Cell harvest and microsome preparation.

At the appropriate time points, cultures of hepatocytes from each treatment group were rinsed twice with ice-cold phosphate-buffered saline. Homogenization buffer (50 mM Tris-HCl, pH 7.0, 150 mM KCl, 2 mM EDTA; 0.5 ml/dish) was added to each dish and cells were scraped with a rubber policeman. Harvested cells from each treatment were then pooled and sonicated with a Vibra-Cell probe sonicator (Sonics & Materials, Danbury, CT) at 40 W for 15 to 20 s (Wortelboer et al., 1990). Cell lysates were centrifuged at 9000g for 20 min at 4°C. Supernatant fractions were collected and centrifuged at 100,000g for 60 min at 4°C. The final microsomal pellets were resuspended in 0.25 to 0.5 ml of 0.25 M sucrose with the aid of a Dounce homogenizer fitted with a Teflon pestle. A small aliquot was taken for protein determination and all samples were subsequently stored at −80°C.

Protein determination.

The concentration of protein was determined with a commercially available kit (BCA Protein Assay kit; Pierce Chemical Co., Rockford, IL) with bovine serum albumin as a standard (supplied with the BCA Protein Assay kit).

P-450 enzyme activities.

7-Ethoxyresorufin and 7-pentoxyresorufin O-dealkylase (PROD), testosterone, and lauric acid hydroxylase activities were determined as described by Pearce et al. (1996).

SDS-polyacrylamide gel electrophoresis and Western immunoblotting.

The levels of CYP2B in rat hepatocyte microsomes were determined by Western immunoblot analysis with polyclonal antibodies against purified rat liver microsomal enzymes as described previously (Parkinson and Gemzik, 1991).

Electron microscopy.

Hepatocyte cultures were fixed with 2.5% glutaraldehyde in 0.1 M phosphate buffer, pH 7.2, for 20 to 30 min. Cultures were then postfixed with 1% OsO4 for 30 min, stained en bloc with 1% uranyl acetate, dehydrated in an acetone series, and embedded in Epon resin. Thin sections were cut perpendicular to the plane of the monolayer, poststained with uranyl acetate and lead citrate, and viewed with a JEOL 1200EXII electron microscope.

Statistical analysis.

Data were analyzed for statistically significant differences between control and treated rat hepatocyte cultures by Dunnett’s two-way ANOVA at the 5% level of significance (Dunnett, 1964).

Results

Media and Overlay Effects on P-450 Enzyme Induction.

Hepatocytes were cultured on a simple substratum of collagen, type I, with one of four different medium formulations. In addition, cultures were overlaid with a top layer of either collagen (type I) or Matrigel. Hepatocyte cultures were then examined for the induction of CYP2B by the prototypical inducer PB. The effects of medium formulation and matrix overlay on microsomal PROD activity in primary cultures of rat hepatocytes treated with 100 μM PB are shown in Fig.1A. WEM was apparently superior to other medium formulations in terms of the magnitude of CYP2B induction (on a per mg microsomal protein basis). MCM and HCM were similar in their capacity to sustain induction of CYP2B by PB, and DMEM was only slightly less effective when hepatocytes were maintained on Permanox dishes. The changes in PROD activity observed in cultures maintained in different media were also reflected qualitatively by marked increases in the immunoreactive protein on Western blots (Fig. 1B).

Figure 1
Figure 1

Effects of medium formulation and extracellular matrix overlay on the induction of PROD activity and immunoreactive CYP2B protein by PB in rat hepatocytes cultured on Permanox dishes.

C, collagen; M, Matrigel; Con, control; PB, 100 μM PB; 2B1, 2 pmol of purified CYP2B1. Data represent the mean ± S.D. of three to four separate experiments.

When hepatocytes were cultured on polystyrene dishes (LUX), WEM again supported the highest degree of CYP2B induction, and MCM and HCM were only slightly less effective (Fig. 2). However, induction of CYP2B by PB was ∼50% lower in cultures maintained on polystyrene dishes than in those on Permanox dishes. Moreover, hepatocytes maintained on polystyrene dishes in DMEM responded only poorly to PB treatment compared with those maintained on Permanox dishes. Regardless of the type of Petri dish used for the experiments, no obvious improvement could be discerned in the induction of CYP2B activity by cultures maintained with a Matrigel versus a collagen overlay. However, all conditions were superior to the induction of CYP2B by PB observed in cultures of hepatocytes without an overlay (data not shown; Sidhu et al., 1993; LeCluyse et al., 1996a).

Figure 2
Figure 2

Effects of medium formulation and extracellular matrix overlay on the induction of PROD activity (CYP2B) by PB in rat hepatocytes cultured on polystyrene (LUX) dishes.

Data represent the average of two separate experiments.

Although the data suggest that WEM facilitates the highest overall induction of CYP2B by PB, other cellular functions were expressed poorly in cultures maintained in WEM compared with those maintained in MCM. For example, medium formulation affected the yield of microsomal protein per dish as shown in Fig. 3. Protein yields were often 1.5 to 2.0 times greater from cultures maintained in enriched media, such as MCM, compared with those from cultures maintained in WEM. The effects of medium formulation on cell morphology and overall monolayer integrity are shown in Figs.4 and 5. Hepatocyte monolayers maintained in MCM are considerably more cuboidal and three-dimensional in shape as indicated by the difficulty of obtaining a uniform plane of focus (Fig. 4). Ultrastructural analysis by electron microscopy revealed that hepatocyte monolayers maintained in MCM exhibit a more normal cuboidal shape and cellular architecture (Fig. 5A). Bile canaliculi were readily apparent at the midplane of the lateral membranes and exhibited normal ultrastructural features such as microvilli and junctional complexes. Hepatocytes cultured in MCM possessed one or two centrally located nuclei and mitochondria, and glycogen granules were more uniformly distributed compared with hepatocytes cultured in WEM. Golgi complexes were localized near bile canaliculi similar to their distribution in vivo. Conversely, hepatocytes maintained in WEM appeared more spread and flattened with displaced ultrastructural features (Fig. 5B).

Figure 3
Figure 3

The effects of medium formulation on the amount of microsomal protein per dish obtained from primary cultures of rat hepatocytes.

Hepatocytes were maintained in WEM or MCM for 3 days without treatment followed by daily treatment with 100 μM PB or solvent alone (controls) for an additional 3 days. The total amount of microsomal protein from each treatment group was determined in the final suspensions and divided by the number of dishes harvested from each group. The data represent the mean ± S.D. from three to four separate experiments.

Figure 4
Figure 4

Phase-contrast light micrographs of rat hepatocyte cultures maintained in either MCM (A) or WEM (B).

Primary cultures of rat hepatocytes were prepared as detailed inExperimental Procedures, and plated onto collagen-coated Permanox dishes and overlaid with Matrigel. Note that hepatocytes maintained in either medium condition establish chord-like arrays with uncovered spaces in between. Hepatocytes maintained in MCM exhibit a more three-dimensional appearance as evidenced by the difficulty in acquiring an even plane of focus throughout the field. Magnification: 750×.

Figure 5
Figure 5

Electron micrographs of rat hepatocyte cultures maintained in either MCM (A) or WEM (B).

Hepatocytes in MCM (A) exhibit a more normal cuboidal shape, a uniform distribution of organelles, a centrally located nucleus, and bile canaliculi located at the midplane of the lateral membranes. By contrast, hepatocytes cultured in WEM (B) exhibit a more flattened shape, abnormal organization of the cytoplasmic elements, and displaced canalicular structures. BC, bile canaliculus; mg, Matrigel overlay; N, nucleus; M, mitochondrion. Bar = 5 μm.

Concentration-Response Relation for P-450 Induction by PB.

The effects of PB concentration on the induction of PROD activity in cultures of hepatocytes maintained in either WEM or MCM are shown in Fig. 6A. A concentration-dependent increase in microsomal PROD activity was observed between 10 and 250 μM PB in cultures of hepatocytes maintained in either medium. At higher concentrations, PB caused a marked reduction in PROD activity. In contrast to CYP2B activity, CYP1A (7-ethoxyresorufinO-dealkylase) and CYP3A (testosterone 6β-hydroxylase) activities did not increase significantly over control levels at concentrations of PB below 250 μM, but increased in a concentration-dependent fashion at higher doses of PB (data not shown).

Figure 6
Figure 6

Concentration-response relationship for induction of microsomal PROD activity (CYP2B) by PB in primary cultures of rat hepatocytes maintained in either WEM or MCM.

Hepatocytes were maintained for 3 days in culture without treatment followed by 3 consecutive days of treatment with 10, 25, 100, 250, 750, or 1500 μM PB. Hepatocytes from each treatment group were then harvested and microsomes were prepared as outlined inExperimental Procedures.

Time Course of P-450 Induction Response.

The effects of treatment time on CYP2B enzyme induction by PB (100 μM) are shown in Fig. 7. PB caused a time-dependent increase in the rate of PROD over the first 72 h of treatment. However, PROD activity did not appear to be increased further by an additional 24 h of treatment with the inducer. The increase in PROD activity observed over the first 72 h was paralleled by a corresponding increase in immunoreactive CYP2B protein (Fig. 7, top).

Figure 7
Figure 7

Time course of induction of microsomal PROD activity and immunoreactive CYP2B protein by PB (100 μM) in rat hepatocytes cultured on collagen-coated Permanox dishes with a Matrigel overlay in modified MCM.

Hepatocytes were maintained for 3 days in culture without treatment followed by treatment with 100 μM PB for 24, 48, 72, or 96 h. The data represent the mean ± S.E. from three separate experiments. *Significantly different from each other according to Dunnett’s test at the 5% level of significance (p = .05). All values were significantly different from controls.

Induction of Other Major P-450 Subfamilies In Vitro.

The culture conditions found to be both optimal and the most time-effective for assessing the induction of CYP2B enzymes, namely MCM, collagen-coated Permanox dishes, and a Matrigel overlay, were utilized to examine the induction of other P-450 enzymes. Figure8 shows the results of three separate experiments designed to examine the induction of CYP1A, CYP2B, CYP3A, and CYP4A by β-naphthoflavone, PB, dexamethasone, and clofibric acid in primary cultures of rat hepatocytes. Induction of rat CYP1A, CYP2B, CYP3A, and CYP4A enzymes was determined in microsomes prepared from three separate groups of hepatocytes, based on measurements of 7-ethoxyresorufin O-dealkylase, PROD, testosterone 6β-hydroxylation, and lauric acid 12-hydroxylation, respectively (Pearce et al., 1996). The corresponding means ± S.E. for each group are compared with representative values obtained from liver microsomes from male rats treated with P-450 enzyme inducers in Table1. The results showed that the culture conditions found to be optimal for CYP2B induction supported the induction of CYP1A, CYP3A, and CYP4A as well. In addition, induction of these P-450 enzymes in cultured rat hepatocytes was reproducible from one experiment to the next and, generally, within a factor of 2 to 4 of induced activities observed in vivo, with the exception of the induction of CYP1A by β-naphthoflavone (Table 1).

Figure 8
Figure 8

Extent and reproducibility of the induction of CYP1A (A), CYP2B (B), CYP3A (C), and CYP4A (D) enzyme activities by prototypical inducers in primary cultures of rat hepatocytes.

Hepatocytes were isolated from three separate livers and cultured on collagen-coated Permanox dishes with a Matrigel overlay in MCM. Hepatocyte cultures were treated for 3 consecutive days with solvent alone (control), or with 50 μM β-naphthoflavone, 100 μM PB, 10 μM dexamethasone, or 100 μM clofibric acid, as indicated in the figure. Microsomes were subsequently prepared and analyzed for CYP1A, CYP2B, CYP3A, and CYP4A enzyme activities as described inExperimental Procedures.

View this table:
Table 1

Comparison of in vivo and in vitro induction of rat liver CYP enzymes by prototypical inducers

Discussion

Media and Overlay Effects on P-450 Enzyme Induction.

The aim of this study was to examine in vitro conditions for evaluating drugs and other xenobiotics as CYP inducers in primary cultures of rat hepatocytes. Effects of media formulation and extracellular matrix overlay were assessed on CYP induction by prototypical inducers in rat hepatocyte cultures to determine the optimum response in vitro. CYP2B induction was chosen as the target response because it is one of the most liver-selective in its regulation and has historically been one of the most difficult responses to restore in primary cultures of rat hepatocytes (LeCluyse et al., 1996b). Four medium formulations, namely, DMEM, WEM, MCM, and HCM, were chosen to represent both standard formulations, which are easily obtained commercially (WEM, DMEM) and enriched formulations, which contain superphysiological concentrations of certain components, especially amino acids (MCM, HCM; LeCluyse et al., 1996b).

The results of this study showed that WEM was superior to other medium formulations in terms of the magnitude of CYP2B induction and specific content of CYP2B in microsomes. MCM and HCM were intermediate in their capacity to sustain induction of CYP2B by PB, and DMEM was slightly less effective when hepatocytes were maintained on Permanox dishes. However, the enriched culture media (MCM, HCM) were superior in terms of maintaining the best overall performance of the cultures, such as cellular integrity, microsomal protein yields, and P-450 induction. The greater induction of CYP2B activity and immunoreactive protein observed in cultures of hepatocytes maintained in WEM may be due, in part, to the down-regulation of other cellular and microsomal proteins, as indicated by the reduction in total microsomal protein per dish. The result of down-regulating other proteins would, in effect, artificially enhance the specific content of CYP2B proteins. On the other hand, cultures of hepatocytes maintained in enriched media have been shown to sustain greater overall levels of protein synthesis, to remain viable for greater lengths of time in culture, to restore carboxyesterase expression and secretion, and to exhibit more normal ultrastructural features compared with other media, such as WEM (Jauregui et al., 1986;Yan et al., 1995; LeCluyse et al., 1996b).

Overall induction of CYP2B by PB was, on average, 50% lower in cultures maintained on LUX dishes compared with those on Permanox dishes. In addition, hepatocytes maintained on polystyrene dishes in DMEM responded poorly to PB treatment in comparison to those maintained on Permanox dishes. The difference in CYP2B induction response between hepatocytes cultured on different types of Petri dishes is not understood at this point but does not appear to be related to differences in the viability and/or integrity of the cells based on trypan blue exclusion and lactate dehydrogenase release (results not shown).

Throughout this study, a matrix overlay was used because it has been reported to enhance P-450 enzyme induction by prototypical inducers, such as PB, and to facilitate a more normal cellular architecture (Musat et al., 1993; Sidhu et al., 1993, 1994; LeCluyse et al., 1994). Matrigel was compared with collagen (type I) as the overlay matrix because both extracellular matrices have been shown to enhance the performance of hepatocyte cultures and to prolong viability, yet no side-by-side comparisons have been reported. Matrigel is more expensive than collagen, type I (per milligram basis), and its composition is complex and variable. However, Matrigel is believed to be superior to collagen in its ability to restore the differentiated phenotype to cultured hepatocytes, presumably due to its complex nature and the apparent requirement for laminin to maintain the PB-mediated CYP2B induction response (Kleinman et al., 1982, 1986; Brown et al., 1995).

Although previous reports have shown the importance of the extracellular matrix environment for restoring the PB-mediated induction of CYP2B enzymes in vitro (Sidhu et al., 1993, 1994), our results showed that little or no difference in the induction of CYP2B by PB was observed between hepatocytes overlaid with collagen and those overlaid with Matrigel. These data suggest that the type of extracellular matrix material used for overlaying hepatocyte monolayers is not crucial, but merely the presence of an overlay is sufficient for restoring P-450 enzyme induction response. These results are in good agreement with those of Dunn et al. (1991), who showed that albumin production and secretion were not dependent on the composition of the overlay matrix. However, Brown et al. (1995) showed that purified laminin (the major component of Matrigel) alone or peptides known to mimic the activities of laminin were sufficient to restore the PB responsiveness to cultured rat hepatocytes maintained on collagen-coated dishes. Aside from the specific mechanism of action, overlaying monolayers with Matrigel holds the distinct advantage of saving time and is easier to perform compared with overlaying monolayers with neutralized collagen (Vitrogen). Therefore, overlaying with Matrigel became the method of choice for subsequent studies.

PB Concentration Response.

The importance of drug concentration is illustrated in Fig. 6, which shows the enzyme-inducing effects of a wide range of concentrations of PB in cultured rat hepatocytes. Over the range of 10 to 100 μM, PB caused a concentration-dependent induction of PROD activity. A plateau was observed between 100 and 300 μM (a range that compares favorably with plasma concentrations in rats administered PB at sedating dosages of 80–100 mg/kg, which is the dosage range typically used to achieve maximal CYP2B induction in vivo; Madan et al., 1999). At concentrations greater than 300 μM, the ability of PB to induce CYP2B activity steadily declined (as did the levels of immunoreactive protein), such that millimolar concentrations of PB were no more effective than 10 μM PB at inducing CYP2B enzymes. The apparent loss of CYP2B induction at high dosages of PB was not a consequence of cell toxicity, and, indeed, millimolar concentrations of PB were extremely effective at inducing CYP1A1, a phenomenon that is not observed in vivo (arguably because millimolar concentrations of PB cannot be achieved in vivo), but that has been reported as an in vitro phenomenon by other investigators (Sidhu et al., 1993). A practical consequence of the bell-shaped curve shown in Fig. 6 is that new chemical entities should be examined for their ability to induce CYP at multiple concentrations, preferably over a range that is clinically and/or toxicologically relevant.

In Vitro Prediction of P-450 Induction.

The results from these studies emphasize three important features of the rat hepatocyte culture technique for measuring P-450 enzyme induction. First, overall, the rat hepatocyte technique for measuring P-450 enzyme induction in vitro is suitably reproducible. Second, the in vitro technique provides a sensitive method for detecting P-450 enzyme induction because the control enzyme activities are lower than those observed in vivo, whereas the induced enzyme activities for CYP2B, CYP3A, and CYP4A are comparable (within a factor of 2–4) to those observed in vivo (Table 1; Parkinson, 1996). By contrast, the induced values for CYP1A in vitro were only within a factor of ∼7 to those observed in vivo, yet the fold induction observed for CYP1A was essentially the same in vivo and in vitro. It is clear from a comparison of in vitro and ex vivo data that induction of P-450 activity in cultured rat hepatocytes does not simply involve restoring activity to the original control level, an observation that was confirmed by Western immunoblotting. Third, cultured rat hepatocytes responded to all four types of P-450 inducers examined. In this context, it is noteworthy that we place considerable emphasis on the response elicited by PB. Induction of CYP2B enzymes by PB is the least robust of the P-450 induction responses depicted in Fig. 8. As a rule of thumb, hepatocytes that respond well to PB will respond to the other types of P-450 inducers. In contrast, it is possible for hepatocytes to respond to CYP1A inducers (as do most extrahepatic tissues and several immortalized cell lines) and yet not respond to PB (which fails to induce in most extrahepatic tissues and immortalized cell lines, suggesting that induction of CYP2B enzymes is highly dependent on the expression of certain liver-specific genes).

In conclusion, when rat hepatocytes are cultured under conditions that restore near normal morphology and expression of liver-specific genes, CYP enzymes can be induced in vitro to levels that are comparable to those achieved in vivo. This in vitro technique provides a sensitive method for evaluating the ability of drugs to induce microsomal P-450 enzymes and can be used as an initial screen for P-450 induction if attention is paid to the potential for false negative and false positive results. The technique would appear appropriate for screening back-up compounds or possibly screening combinatorial libraries, where the risk of false results can be arguably offset by the benefit of high throughput. Primary cultures of hepatocytes would be particularly useful for comparing the enzyme-inducing capabilities of a racemic mixture with that of its individual enantiomers, or evaluating the enzyme-inducing potential of any compound whose supply is too limited to permit an ex vivo analysis of enzyme induction. Thus, primary cultures of rat hepatocytes maintained under appropriate culture conditions should serve as a useful tool in preclinical drug development as a screen for inducers of P-450 enzyme activity.

Acknowledgments

We thank Rick Gould, Kenda Marcucci, Jason Latham, Kevin Smith, D. James Coon, and Scott Barros for technical assistance.

Footnotes

  • Send reprint requests to: Dr. Ajay Madan, XenoTech, LLC, 3800 Cambridge, Kansas City, KS 66103.

  • ↵1 Present address: School of Pharmacy, Division of Drug Delivery and Disposition, CB# 7360, Beard Hall, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599.

  • ↵2 Present address: Cerep, Inc., 15318 NE 95th St., Redmond, WA 98052.

  • This work was funded in part by National Institutes of Health Grants ES-03765 and GM-37044, Du Pont Pharmaceuticals Company, Pfizer, Inc., and the Procter and Gamble International Program for Animal Alternatives.

  • Abbreviations used are::
    CYP
    cytochrome P-450 enzymes
    DMEM
    Dulbecco’s modified Eagle’s medium
    HCM
    hepatocyte culture medium
    ITS+
    insulin, transferrin, selenium, linoleic acid, and BSA supplement
    MCM
    modified Chee’s medium
    PROD
    7-pentoxyresorufin O-dealkylase
    WEM
    Williams’ E medium
    PB
    phenobarbital
    • Received January 22, 1999.
    • Accepted April 27, 1999.

References

  1. ↵
    1. Adesnik M,
    2. Bar-Nun S,
    3. Maschio F,
    4. Zunich M,
    5. Lippman A,
    6. Bard E
    (1981) Mechanism of induction of cytochrome P-450 by phenobarbital. J Biol Chem 256:10340–10345.
    OpenUrlAbstract/FREE Full Text
  2. ↵
    1. Ben-Ze’ev A,
    2. Robinson GS,
    3. Bucher NL,
    4. Farmer SR
    (1988) Cell-cell and cell-matrix interactions differentially regulate the expression of hepatic and cytoskeletal genes in primary cultures of rat hepatocytes. Proc Natl Acad Sci USA 85:2161–2165.
    OpenUrlAbstract/FREE Full Text
  3. ↵
    1. Berry MN,
    2. Edwards AM,
    3. Barritt GJ
    (1991) Isolated Hepatocytes: Preparation, Properties and Applications. (Elsevier Science Publishing Co. New York).
  4. ↵
    1. Brown SE,
    2. Guzelian CP,
    3. Schuetz E,
    4. Quattrochi LC,
    5. Kleinman HK,
    6. Guzelian PS
    (1995) Critical role of extracellular matrix on induction by phenobarbital of cytochrome P450 2B1/2 in primary cultures of adult rat hepatocytes. Lab Invest 73:818–827.
    OpenUrlPubMed
  5. ↵
    1. Dahn MS,
    2. Hsu CJ,
    3. Lange MP,
    4. Kimball SR,
    5. Jefferson LS
    (1993) Factors affecting secretory protein production in primary cultures of rat hepatocytes. Proc Soc Exp Biol Med 203:38–44.
    OpenUrlAbstract/FREE Full Text
  6. ↵
    1. Dich J,
    2. Vind C,
    3. Grunnet N
    (1988) Long-term culture of hepatocytes. Effect of hormones on enzyme activities and metabolic capacity. Hepatology 8:39–45.
    OpenUrlPubMed
  7. ↵
    1. Dunn JCY,
    2. Tompkins RG,
    3. Yarmush ML
    (1991) Long-term in vitro function of adult hepatocytes in a collagen sandwich configuration. Biotechnol Prog 7:237–245.
    OpenUrlCrossRefPubMed
  8. ↵
    1. Dunnett GW
    (1964) New tables for multiple comparisons with a control. Biometrics 20:482–491.
    OpenUrlCrossRef
  9. ↵
    1. Jauregui HO,
    2. McMillan PN,
    3. Driscoll J,
    4. Naik S
    (1986) Attachment and long term survival of adult rat hepatocytes in primary monolayer cultures: Comparison of different substrata and tissue culture media formulations. In Vitro Cell Dev Biol 22:13–22.
    OpenUrlPubMed
  10. ↵
    1. Kleinman HK,
    2. McGarvey ML,
    3. Hassell JR,
    4. Star VL,
    5. Cannon FB,
    6. Laurie GW,
    7. Martin GR
    (1986) Basement membrane complexes with biological activity. Biochemistry 25:312–318.
    OpenUrlCrossRefPubMed
  11. ↵
    1. Kleinman HK,
    2. McGarvey ML,
    3. Liotta LA,
    4. Robey PG,
    5. Tryggvason K,
    6. Martin GR
    (1982) Isolation and characterization of type IV procollagen, laminin and heparan sulfate proteoglycan from the EHS sarcoma. Biochemistry 21:6188–6193.
    OpenUrlCrossRefPubMed
  12. ↵
    1. LeCluyse EL,
    2. Audus KL,
    3. Hochman JH
    (1994) Formation of extensive canalicular networks by rat hepatocytes cultured in collagen-sandwich configuration. Am J Physiol 266:C1764–C1774.
    OpenUrlAbstract/FREE Full Text
  13. ↵
    1. Borchard RT,
    2. Smith PL,
    3. Wilson G
    1. LeCluyse EL,
    2. Bullock P,
    3. Parkinson A,
    4. Hochman J
    (1996a) Cultured rat hepatocytes. in Models for Assessing Drug Absorption and Metabolism, eds Borchard RT, Smith PL, Wilson G (Plenum Press, New York), pp 121–160.
  14. ↵
    1. LeCluyse EL,
    2. Bullock P,
    3. Parkinson A
    (1996b) Strategies for restoration and maintenance of normal hepatic structure and function in long-term cultures of rat hepatocytes. Adv Drug Delivery Rev 22:133–186.
    OpenUrlCrossRef
  15. ↵
    1. Lindblad WJ,
    2. Schuetz EG,
    3. Redford KS,
    4. Guzelian PS
    (1991) Hepatocellular phenotype in vitro is influenced by biophysical features of the collagenous substratum. Hepatology 13:282–288.
    OpenUrlCrossRefPubMed
  16. ↵
    1. Madan A,
    2. De Haan R,
    3. Mudra D,
    4. Carroll K,
    5. LeCluyse E,
    6. Parkinson A
    (1999) Effect of cryopreservation on cytochrome P450 enzyme induction in cultured rat hepatocytes. Drug Metab Dispos 27:327–335.
    OpenUrlAbstract/FREE Full Text
  17. ↵
    1. Musat AI,
    2. Sattler C,
    3. Sattler GL,
    4. Pitot HC
    (1993) Re-establishment of cell polarity of hepatocytes in primary culture. Hepatology 18:198–205.
    OpenUrlCrossRefPubMed
  18. ↵
    1. Klaassen CD
    1. Parkinson A
    (1996) Biotransformation of xenobiotics. in Casarett and Doull’s Toxicology. The Basic Science of Poisons, ed Klaassen CD (McGraw-Hill, New York), 5th ed, pp 113–186.
  19. ↵
    1. Parkinson A,
    2. Gemzik B
    (1991) Production and purification of antibodies against rat liver P450 enzymes. Methods Enzymol 206:233–245.
    OpenUrlPubMed
  20. ↵
    1. Pearce RE,
    2. McIntyre CJ,
    3. Madan A,
    4. Sanzgiri U,
    5. Draper AJ,
    6. Bullock PL,
    7. Cook DC,
    8. Burton LA,
    9. Latham J,
    10. Nevins C,
    11. Parkinson A
    (1996) Effects of freezing, thawing, and storing human liver microsomes on cytochrome P450 activity. Arch Biochem Biophys 331:145–169.
    OpenUrlCrossRefPubMed
  21. ↵
    1. Pollard JW,
    2. Walker JM
    1. Quistorff B,
    2. Dich J,
    3. Grunnet N
    (1989) Preparation of isolated rat liver hepatocytes. in Methods in Molecular Biology, eds Pollard JW, Walker JM (Humana Press, Clifton, NJ), 5: Animal Cell Culture:151–160.
  22. ↵
    1. Reid LM,
    2. Fiorino AS,
    3. Sigal SH,
    4. Brill S,
    5. Holst PA
    (1992) Extracellular matrix gradients in the space of Disse: Relevance to liver biology. Hepatology 15:1198–1203.
    OpenUrlCrossRefPubMed
  23. ↵
    1. Ryan DE,
    2. Levin W
    (1990) Purification and characterization of hepatic microsomal cytochrome P-450. Pharmacol Ther 45:153–239.
    OpenUrlCrossRefPubMed
  24. ↵
    1. Sidhu JS,
    2. Farin FM,
    3. Kavanagh TJ,
    4. Omiecinski CJ
    (1994) Effect of tissue-culture substratum and extracellular matrix overlay on liver-selective and xenobiotic inducible gene expression in primary rat hepatocytes. In Vitro Toxicol 7:225–242.
    OpenUrl
  25. ↵
    1. Sidhu JS,
    2. Farin FM,
    3. Omiecinski CJ
    (1993) Influence of extracellular matrix overlay on phenobarbital-mediated induction of CYP2B1, 2B2, and 3A1 genes in primary adult rat hepatocyte culture. Arch Biochem Biophys 30:103–113.
    OpenUrl
  26. ↵
    1. Sonderfan AJ,
    2. Arlotto MP,
    3. Dutton DR,
    4. McMillen SK,
    5. Parkinson A
    (1987) Regulation of testosterone hydroxylation by rat liver microsomal cytochrome P-450. Arch Biochem Biophys 255:27–41.
    OpenUrlCrossRefPubMed
  27. ↵
    1. Thomas PE,
    2. Bandiera S,
    3. Reik LM,
    4. Maines SL,
    5. Ryan DE,
    6. Levin W
    (1987) Polyclonal and monoclonal antibodies as probes of rat hepatic cytochrome P-450 isozymes. Fed Proc 46:2563–2566.
    OpenUrlPubMed
  28. ↵
    1. Turner NA,
    2. Pitot HC
    (1989) Dependence of the induction of cytochrome P-450 by phenobarbital in primary cultures of adult rat hepatocytes on the composition of the culture medium. Biochem Pharmacol 38:2247–2251.
    OpenUrlCrossRefPubMed
  29. ↵
    1. Waxman DJ,
    2. Morrissey JJ,
    3. Naik S,
    4. Jauregui HO
    (1990) Phenobarbital induction of cytochromes P450: High-level long-term responsiveness of primary rat hepatocyte cultures to drug induction, and glucocorticoid dependence of the phenobarbital response. Biochem J 271:113–119.
    OpenUrlAbstract/FREE Full Text
  30. ↵
    1. Wortelboer HM,
    2. de Kruif C A,
    3. van Iersel AAJ,
    4. Falke HE,
    5. Noordhoek J,
    6. Blaauboer BJ
    (1990) The isoenzyme pattern of cytochrome P450 in rat hepatocytes in primary culture, comparing different enzyme activities in microsomal incubations and in intact monolayers. Biochem Pharmacol 40:2525–2534.
    OpenUrlCrossRefPubMed
  31. ↵
    1. Yan B,
    2. Yang D,
    3. Bullock P,
    4. Parkinson A
    (1995) Rat serum carboxylesterase: Cloning, expression, regulation, and evidence of secretion from liver. J Biol Chem 270:19128–19134.
    OpenUrlAbstract/FREE Full Text
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Influence of Extracellular Matrix Overlay and Medium Formulation on the Induction of Cytochrome P-450 2B Enzymes in Primary Cultures of Rat Hepatocytes

Edward LeCluyse, Peter Bullock, Ajay Madan, Kathy Carroll and Andrew Parkinson
Drug Metabolism and Disposition August 1, 1999, 27 (8) 909-915;
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