Evaluation of the HC-04 cell line as an in vitro model for mechanistic assessment of changes in hepatic cytochrome P450 3A during adenovirus infection

Abstract

HC-04 cells were evaluated as an in vitro model for mechanistic study of changes in the function of hepatic CYP3A during virus infection. Similar to in vivo observations, infection with a first generation recombinant adenovirus significantly inhibited CYP3A4 catalytic activity in an isoform-specific manner. Virus (MOI 100) significantly reduced expression of the retinoid X receptor (RXR) by 30% 96 hours after infection. Cytoplasmic concentrations of the pregnane X receptor (PXR) were reduced by 50%, whereas the amount of the constitutive androstane receptor (CAR) in the nuclear fraction doubled with respect to uninfected controls. Hepatocyte nuclear factor 4α (HNF-4α) and peroxisome proliferator-activated receptor γ coactivator 1α (PGC-1α) were also reduced by ∼70% during infection. Virus suppressed CYP3A4 activity in the presence of the PXR agonist rifampicin and did not affect CYP3A4 activity in the presence of the CAR agonist CITCO [6-(4-chlorophenyl) imidazo[2,1-b][1,3]thiazole-5-carbaldehyde-O-(3,4-dichlorobenzyl)oxime], suggesting that virus-induced modification of PXR may be responsible for observed changes in hepatic CYP3A4. The HC-04 cell line is easy to maintain, and CYP3A4 in these cells was responsive to known inducers and suppressors. Dexamethasone (200 μM) and phenobarbital (500 μM) increased activity by 230 and 124%, whereas ketoconazole (10 μM) and lipopolysaccharide (LPS) (10 μg/ml) reduced activity by 90 and 92%, respectively. This suggests that HC-04 cells can be a valuable tool for mechanistic study of drug metabolism during infection and for routine toxicological screening of novel compounds prior to use in the clinic.

Fig. 1.

Primary hepatocytes isolated from male Sprague-Dawley rats respond to adenovirus infection in a manner similar to that observed in vivo when seeded on standard tissue culture dishes. (A) A first generation adenovirus containing a beta-galactosidase transgene (AdlacZ) did not significantly alter CYP3A activity of primary hepatocytes sandwiched between a collagen matrix. (B) Histochemical stain for the beta-galactosidase transgene indicates that the sandwich matrix prevented virus infection. (C) CYP3A activity of primary hepatocytes cultured on standard untreated tissue culture dishes responds to virus infection in a dose-dependent manner (MOI, multiplicity of infection). (D) Representative images of hepatocytes after histochemical staining for endogenous peroxidase activity before and after removal of Kupffer Cells (KC) through magnetic bead separation. Cells containing a black/blue precipitate exhibited endogenous peroxidase activity, a characteristic unique to KCs and absent in hepatocytes (Widmann et al., 1972). In each panel, cells were seeded at a density of 5 × 10⁵ cells/mm. (A and C) (n = 6 replicates/group) *P ≤ 0.05, **P ≤ 0.01, Bonferroni Dunn post hoc test. Magnification: 100× (B), 400× (D).

Fig. 2.

HC-04 cells respond to known CYP3A4 suppressors and respond to adenovirus infection in a CYP3A4 isoform-specific manner. (A) Substances known to suppress CYP3A4 activity were added to culture media daily over a period of 3 days. Ketocon, ketoconazole; Erythro, erythromycin; LPS, bacterial lipopolysaccharides; CyA, cyclosporine A. (B) Cells were infected with 500 MOI AdlacZ over time. In vitro catalytic activity was measured by incubation of infected cultures with testosterone for 30 minutes and quantitation of the CYP3A isoform-specific metabolite, 6β-hydroxytestosterone by HPLC. (C) Cells were treated as described for (B) except that the CYP2D-specific testosterone metabolite 16α-hydroxytestosterone was measured by HPLC. In each panel, results are reported as the mean ± standard error of the mean of data generated from three 100 mm culture plates per condition replicated in three separate experiments. Statistical significance was determined between individual treatment groups and saline-treated controls by one-way analysis of variance with a Bonferroni/Dunn post hoc test. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001.

Fig. 3.

Adenovirus infection inhibits CYP3A activity and mRNA levels in HC-04 cells. (A) Catalytic activity of CYP3A in HC-04 cells after virus infection. Activity was measured by quantitation of the CYP3A-specific testosterone metabolite 6β-hydroxytestosterone by HPLC. (B) CYP3A4 expression and representative gel of RT PCR products. cDNA was amplified under the following conditions: 95°C for 45 seconds, 57°C for 45 seconds, and 75°C for 1 minute for 28 cycles. Data are reported as the ratio of band intensity of CYP3A4 with respect to 18S with the ratio for uninfected controls (MOI 0) normalized to 1. (C) Immunoblot analysis of CYP3A4 protein. A representative blot illustrating band intensity for each virus concentration is included under the plot summarizing the change in CYP3A4 protein concentration with respect to beta-actin. Data in each panel was collected 96 hours after virus infection. Data are reported as the mean ± standard error of the mean of three 100 mm culture plates per condition from three separate experiments. Statistical significance was determined between data generated from individual treatment groups and uninfected (MOI 0) controls by one-way analysis of variance with a Bonferroni/Dunn post-hoc test. **P < 0.01 and ***P < 0.001.

Fig. 4.

Transgene expression patterns in cultured cells 48 hours after infection. Cells were infected with a first generation recombinant adenovirus expressing beta-galactosidase (AdlacZ) at MOIs of 0 (A and D), 500 (B and E), and 1000 (C and F). Primary rat hepatocytes are shown in (A–C). HC-04 cells are shown in (D–F). Transgene expression in HC-04 cells was highly concentrated throughout the cell, whereas that in primary hepatocytes was concentrated in the nucleus and diffuse throughout the cytoplasm. Uninfected cultures in (A) and (D) received histochemical treatment as did infected cells to detect endogenous levels of beta-galactosidase. None was detected in either cell type. Magnification: 250× (A); 100× (B–F).

Fig. 5.

Adenovirus infection significantly alters nuclear and cytoplasmic levels of the RXR nuclear receptor 96 hours after infection in HC-04 cells. Immunoblot analysis of nuclear (A) and cytoplasmic (B) extracts for RXR protein expression after infection with a first generation recombinant adenovirus. Representative blots illustrating band intensity for each treatment condition are shown under plots. Protein levels are reported in arbitrary units of relative density as compared with a known protein standard. Data are reported as the mean ± standard error of the mean of three 100 mm culture plates per condition collected from three separate experiments. Statistical significance was determined between individual treatment groups and saline-treated controls (MOI 0) by one-way analysis of variance with a Bonferroni/Dunn post hoc test. *P ≤ 0.05, **P ≤ 0.01, and ***P ≤ 0.001.

Fig. 6.

Adenovirus infection significantly alters PXR protein levels in the cytoplasm of HC-04 cells. Immunoblot analysis of nuclear (A) and cytoplasmic (B) extracts for PXR protein expression 96 hours after infection with a first generation recombinant adenovirus (AdlacZ). Representative blots illustrating band intensity for each treatment condition are shown under plots for each virus. Protein levels are reported in arbitrary units of relative density compared with a known protein standard specific for each cellular compartment. Data are reported as the mean ± standard error of the mean of three 100 mm culture plates per condition collected from three separate experiments. Statistical significance was determined between individual treatment groups and saline-treated controls (MOI 0) by one-way analysis of variance with a Bonferroni/Dunn post hoc test. **P ≤ 0.01, and ***P ≤ 0.001.

Fig. 7.

Recombinant adenovirus increases CAR in nuclear extracts of HC-04 cells. Immunoblot analysis of nuclear (A) and cytoplasmic (B) extracts for CAR protein 96 hours after infection with adenovirus. Representative blots illustrating band intensity for each virus concentration are shown. Arrow indicates aberrant band found in samples infected with 500 MOI of virus. Protein levels are reported in arbitrary units of relative density compared with a known protein standard suitable for each cellular compartment. Data are reported as the mean ± standard error of the mean of three 100 mm culture plates per condition collected from three separate experiments. Statistical significance was determined between individual treatment groups and saline-treated controls (MOI 0) by one-way analysis of variance with a Bonferroni/Dunn post hoc test. **P ≤ 0.01.

Fig. 8.

Adenovirus infection reduces HNF-4α and PGC-1α late in the infection process. Twenty micrograms of whole cell lysate was resolved in each lane of a 10% polyacrylamide gel via electrophoresis (SDS-PAGE). Membranes were blocked at room temperature for 2 hours with 3% BSA in Tris-buffered saline containing 0.1% Tween-20 before being probed with one of the following: rabbit anti-PGC-1 (1:1000 dilution, at 4°C overnight), rabbit anti-HNF-α (1:800 dilution, at room temperature for 2 hours), or mouse anti-β-actin (1:1000 dilution, at room temperature for 2 hours). Membranes were then incubated with HRP-conjugated goat anti-rabbit IgG (1:8,000) or HRP-conjugated rabbit anti-mouse IgG (1:15,000) for 1 hour at room temperature before processing. Protein levels are reported in arbitrary units of relative density compared with a known protein standard (β-actin). Representative blots illustrating band intensity for each time point are shown under each plot. Data are reported as the mean ± standard error of the mean of three 100 mm culture plates per condition collected from three separate experiments. Statistical significance was determined between individual treatment groups and uninfected controls (t = 0) by one-way analysis of variance with a Bonferroni/Dunn post hoc test. *P ≤ 0.05.

Fig. 9.

Changes in gene expression patterns induced by virus infection can be detected in HC-04 cells. mRNA levels for each nuclear receptor and representative agarose gels from extracts collected 96 hours after infection with adenovirus at MOIs of 0, 10, 100, and 500. Results are reported as the mean ± standard error of the mean of three 100 mm culture plates per condition collected from three separate experiments. Statistical significance was determined between individual treatment groups and uninfected controls (MOI 0) by one-way analysis of variance with a Bonferroni/Dunn post hoc test. *P ≤ 0.05.

Fig. 10.

Adenovirus overrides the effects of a PXR agonist and suppresses CYP3A activity in HC-04 cells. Stock solutions of rifampicin and CITCO were prepared in DMSO. Compounds were diluted to working concentrations (100 nM CITCO, 20 μM rifampicin) in complete culture media. The final DMSO concentration in these solutions was 0.1%. Cells were treated with each compound for 48 hours prior to infection with adenovirus. Twenty-four hours after infection, CYP3A4 activity was assessed using a P450-Glo CYP3A4 Luciferin-IPA assay kit according to the manufacturer’s instructions. Results are reported as the mean ± standard error of the mean of three culture plates per condition. Statistical significance was determined between individual treatment groups and uninfected controls by one-way analysis of variance with a Bonferroni/Dunn post hoc test. *P ≤ 0.05, **P ≤ 0.01, and ***P ≤ 0.001.