Introduction

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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

Top Abstract Introduction Experimental Procedures Results Discussion References

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%.

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

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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).

View larger version (46K): [in this window] [in a new window]   Fig. 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.

View larger version (38K): [in this window] [in a new window]   Fig. 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.

View larger version (32K): [in this window] [in a new window]   Fig. 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.

View larger version (177K): [in this window] [in a new window]   Fig. 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 in Experimental 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×.

View larger version (122K): [in this window] [in a new window]   Fig. 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-ethoxyresorufin O-dealkylase) and CYP3A (testosterone 6-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).

View larger version (17K): [in this window] [in a new window]   Fig. 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 in Experimental 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).

View larger version (27K): [in this window] [in a new window]   Fig. 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. Figure 8 shows the results of three separate experiments designed to examine the induction of CYP1A, CYP2B, CYP3A, and CYP4A by -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 6-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 Table 1. 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 -naphthoflavone (Table 1).

View larger version (50K): [in this window] [in a new window]   Fig. 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 -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 in Experimental Procedures.

	Discussion

Top Abstract Introduction Experimental Procedures Results Discussion References

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).

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).