ITE was a gift from Dr Jiasheng Song, (AhR Pharmaceuticals, Inc., Madison, WI, USA). Anti-CYP450 isoenzyme 1A1 antibody (ab126828), anti-CYP450 isoenzyme 1B1 antibody (ab33586), anti-CYP450 isoenzyme 1A2 antibody (ab56073), CYP3A4 antibody (ab135813), CYP3A5 antibody (ab108624), CYP2D6 antibody (ab62204), CYP2C9 antibody (ab150364) and CYP2E1 antibody (ab90564) were all purchased from Abcam (Cambridge, MA, USA).

All cell cultures were incubated in a humidified atmosphere at 37°C and 5% CO₂. The Huh7 and C3A cells (CRL-10741; ATCC, Manassas, VA, USA) were cultured in Dulbecco's modified Eagle's medium (DMEM) (12430; Gibco, Auckland, New Zealand) supplemented with 10% fetal bovine serum (FBS) (10099; Gibco Life Technologies, Grand Island, NY, USA) as well as 1% penicillin/streptomycin (Gibco). The cells were detached following incubation with 0.05% trypsin-EDTA (25300; Gibco), counted, and finally diluted to 3×10⁶/ml with 2.0% alginate solution.

ITE was dissolved in DMSO, serial 1,000X stock solutions in DMSO were prepared and 1:1,000 diluted with culture medium into final concentrations of 0.2, 0.5 and 1 μM, respectively. For the 0 μM ITE control group, only the same volume of DMSO was added to the culture medium so that the final concentration of DMSO was also kept at 0.1% (v/v). In most cases, the cells were treated with serially diluted ITE for 24 and 48 h with ITE being replenished everyday.

Alginate encapsulation was performed as previously described (5) with modifications. Briefly, the Huh7 and C3A cells were suspended in 2.0% sodium alginate solution (154 mM NaCl, 10 mM HEPES, pH 7.4). Each mixture was sprayed at a flow rate of 9.5 ml/min through a 300 μm nozzle using an electrostatic microencapsulator unit (Nisco Engineering AG, Zurich, Switzerland). The vibration frequency of the nozzle was kept at 0.30 kHz. The alginate droplets were collected in a calcium chloride gelation bath (154 mM NaCl, 10 mM HEPES, 115 mM CaCl₂, pH 7.4), followed by 10 min of gelling and normal saline washes for three times. Eventually, the Huh7 and the C3A microspheres with the designed 800 μm diameter were produced.

Cell viability was determined by MTT assay (11465007001; Roche Diagnostics, Basel, Switzerland) according to the manufacturer's instructions. Briefly, the cells were cultured with 100 μl of medium per well in 96-well microplates. Following the addition of 10 μl of the MTT labeling reagent (final concentration, 0.5 mg/ml) to each well, the plates were incubated for 4 h in a humidified atmosphere at 37°C and 5% CO₂. The plates were incubated overnight with 100 μl of the solubilization solution in each well. For the formation of purple formazan crystals, proportional to the number of metabolically active viable cells, the absorbance was measured using a microplate reader (DTX880; Beckman Coulter, Inc., Brea, CA, USA) at a wavelength of 570 nm.

The microspheres were washed twice with DMEM medium without phenol red and then stained for 2 min with 5 mg/ml FDA and 10 mg/ml PI in Opti-MEM medium without phenol red (Gibco-Invitrogen Life Technologies, Paisley, Scotland). The stained beads were washed twice with Opti-MEM medium and examined using a Bio-Rad Radiance 2100 confocal microscope (Bio-Rad Laboratories, Inc., Hertfordshire, UK). Images were captured and analyzed using LaserSharp 2000 software (Bio-Rad Laboratories, Inc.).

Various groups of cell culture models were used in our study that included: 2D, cells were cultured in a dish in a routine two-dimensional manner; 3D, cells were encapsulated and cultured under static conditions; and 3D-F, cells were encapsulated and cultured in the fluidized bed bioreactor. CYP450 1A2, 3A4, 1A1 and 1B1 enzyme activity assays were carried out directly in 24-well plates. The measurement of luciferase activity was performed with a P450-Glo CYP1A2 assay (V8422), a CYP3A4 assay (V9002), a CYP1A1 assay (V8752) and a CYP1B1 assay (V8762) (all from Promega, Madison, WI, USA). In brief, the cells were incubated at 37°C in Krebs-Henseleit buffer (K3753; Sigma-Aldrich, St. Louis, MO, USA) containing luciferin-1A2, fresh medium containing luciferin-IPA or luciferin-CEE. After 1 or 4 h of incubation, 50 μl of buffer or culture medium from each well were passed to a 96-well opaque white plate by mixing with an equal volume of the luciferin detection reagent to initiate a luminescent reaction. After 20 min of shaking at room temperature, luminescence was measured using a microplate reader (DTX880; Beckman Coulter, Inc.).

After running the experiments, all media were collected and stored at −80°C. The urea concentration was measured using the urea assay kit (DIUR-500; Bioassay Systems LLC, Hayward, CA, USA). The albumin concentration was assayed with the human albumin ELISA quantitation set (E80-129; Bethyl Laboratories, Montgomery, TX, USA). All results were analyzed with CurveExpert 1.3 software and fitted with logistic model with r2>0.99.

At day 2 of culture, the cells plated in 24-well plates were fixed with 90% ethanol and washed with saline. The fixed cells were incubated with the indicated primary antibodies followed by 2 U/ml of Alexa Fluor^® 488 AffiniPure Rabbit Anti-Goat IgG (H+L) (305-545-003; Jackson ImmunoResearch Laboratories, Inc., West Grove, PA, USA). The nuclei were stained with Hoechst 33342 (Hoechst 33342 Trihydrochloride, Trihydrate - FluoroPure™ Grade, H21492; Thermo Fisher Scientific, Inc., Waltham, MA, USA). The slides were incubated at room temperature in the dark for 20 min, rinsed with saline, and mounted in Vectashield (Vector Laboratories, Peterborough, UK) prior to examination with a confocal microscope.

After washing 3 times with phosphate buffered saline (PBS), the cells were lysed in lysis buffer (no. 9803; Cell Signaling Technology, Inc., Danvers, MA, USA) containing protease inhibitors (Complete Protease Inhibitor Cocktail) and phosphatase inhibitors (PhosSTOP) (both from Roche Diagnostics). Protein concentrations were measured using the BCA assay kit (Thermo Fisher Scientific, Inc.). Proteins in the lysates were separated using SDS-PAGE and immunoblotted with respective antibodies.

The microspheres were dissolved in 55 mM sodium citrate. After washing with PBS twice, RNA was extracted using the RNeasy Mini kit (15596026; Qiagen, Hilden, Germany) according to the manufacturer's instructions. cDNA was synthesized using oligo primers and a reverse transcription kit (037A; both from Takara Bio, Inc., Shiga, Japan). The Bio-Rad Universal SYBR Supermix (72-5121) was used to perform qPCR assays on a Bio-Rad Cycler (C1000) (both from Bio-Rad Laboratories, Inc., Hercules, CA, USA) with various sequences.

Briefly, total RNA was extracted from the C3A cells treated with 0.2 μM ITE using TRIzol reagent (Cat. no. 15596-108; Life Technologies, Carlsbad, CA, USA) following the manufacturer's instructions, and the RIN was tested to inspect RNA integrity using an Agilent Bioanalyzer 2100 (Agilent Technologies, Inc., Santa Clara, CA, USA). Qualified total RNA was further purified using the RNeasy micro kit (Cat. no. 74004) and the RNase-Free DNase Set (Cat. no. 79254) (both from Qiagen). Total RNA was amplified, labeled and purified using the GeneChip 3′ IVT PLUS Reagent kit to obtain biotin-labeled cRNAs (Cat. no. 902416; Affymetrix, Inc., Santa Clara, CA, USA) following the manufacturer's instructions. Array hybridization and washes were performed using the GeneChip^® Hybridization, Wash and Stain kit (Cat. no. 900720) in a hybridization oven 645 (Cat. no. 00-0331-220V) and Fluidics Station 450 (Cat. no. 00-0079) (all from Affymetrix, Inc.) according to the manufacturer's instructions.

Gene expression profiling was conducted by Shanghai Biotechnology Corporation using the Affymetrix PrimeView™ Human Gene Expression Array (Affymetrix, Inc.). All data were analyzed according to the manufacturer's instructions. Raw data originated from Affymetrix CEL files were normalized by RMA background correction and values were log2 transformed. Comparison of the data sets by the t-test showed that 1,472 of the total of 49,293 probe-sets (2.99%) were differentially expressed (|fold changes| ≥2). In order to analyze the enrichment of P-values of each GO term, we used Fisher's exact test to calculate P-values and R package stats to calculate FDR (q-value) by the BH method (www.r-project.org).

Statistical analysis was performed using the Student's t-test and one-way analysis of variance (ANOVA) with SPSS for Windows version 20.0 (SPSS, Inc., Chicago, IL, USA). Data from representative experiments are presented as the means ± standard deviation (SD). Differences were considered statistically significant with a P-value <0.05. The experiments were repeated at least 2 to 3 times in duplicate or triplicate for each condition.

To explore the potential toxicity of ITE on Huh7 and C3A cells cultured in a monolayer, we examined cell viability and morphology in the presence or absence of ITE (Fig. 1). The use of ITE at a concentration of up to 1 μM and treatment for 48 h did not affect the growth of the Huh7 cells. When the C3A cells were treated for 48 h with ITE at a concentration of 0.5 μM and above, ITE slightly yet significantly inhibited the growth of the C3A cells (Fig. 1A). Phase contrast microscopy revealed that treatment with 0.2 μM ITE for 48 h did not have marked effects on the general morphology of either the Huh7 or the C3A cells grown as monolayer cultures (Fig. 1B). As ITE was dissolved in DMSO, the control group (0 μM ITE) control media thus also contained the same quantity of DMSO. Therefore, 0 μM ITE as the control group was selected for the use in the experiments described below.

Following a previous publication (22), we evaluated whether ITE in culture enhances the transcription of several selected metabolism-associated genes, including CYP450 phase I and II enzymes, nuclear receptors and specific proteins. We focused on comparing the expression profiles of Huh7 and C3A cells cultured with ITE versus normal culture (Fig. 2). Our results indicated that the transcription levels of some CYP450-related genes and nuclear receptors were elevated in the cultures containing ITE. After being exposed to ITE, the liver cells may modulate the CYP450-related genes of 1A2, 3A5, UGT1A1, SULT2A1 and nuclear receptors of hHNF-4a in Huh7 cells, and 1A2, 3A4, 3A5, 2B6, 2C8, 2D6, 2E1 and nuclear receptors gene of hHNF-4a in C3A cells.

We then measured the expression of proteins in the CYP450 system in the Huh7 and C3A cells. The CYP1A2, CYP3A4, CYP2E1, CYP1A1 and CYP1B1 protein levels were detected by western blot analysis. The protein levels of CYP3A4, CYP2E1 and CYP1B1 were increased by varying degrees in the presence of 0.2 μM ITE in the culture medium compared to the cells cultured in normal medium; however, CYP1A2 was not affected by ITE treatment (Fig. 3). Amongst the examined CYP450s, the protein levels of CYP3A4 markedly increased in accordance with the metabolic activities (shown below).

Metabolic functions were examined to assess the biotransformation and synthesis capacity of the Huh7 and C3A cells in the presence of ITE in culture (Fig. 4). As expected, the CYP450 activity levels (CYP1A1, CYP1A2, CYP1B1 and CYP3A4) were all significantly increased in the Huh7 and C3A cells cultured with ITE. Of note, 0.2 μM ITE was the optimal concentration for most CYP450s, but the activity of CYP3A4 in the C3A cells was higher at 1 μM ITE (Fig. 4J). The changes in the kinetics of CYP450 activity in response to ITE treatment were different between Huh7 cells and C3A cells (Fig. 4A vs. C and G vs. I). In the Huh7 cells, the activities of both CYP1A1 and CYP1B1 reached the highest levels at 0.2 μM ITE (Fig. 4A and C). By contrast, in the C3A cells (Fig. 4G and I), the activity of CYP1A1 reached the highest level at 0.2 μM ITE and then decreased at 0.5 and 1 μM ITE, while the activity of CYP1B1 kept increasing until 0.5 μM ITE and then started to decrease at 1 μM ITE. Amongst the four CYP450s examined, CYP3A4 exhibited the highest increase in metabolic activity in both cell lines (Fig. 4D and J). The enhancement of CYP450 activity levels indicate a higher detoxification capacity in liver cells maintained in our new culture condition. By contrast, albumin secretion (Fig. 4E and K) and urea synthesis (Fig. 4F and L), were only slightly increased by ITE under these conditions and did not reach statistical significance.

To explore the potential toxic effect of ITE on Huh7 and C3A cells cultured in alginate microspheres, we determined the cellular morphology and viability in the presence or absence of ITE in culture (Fig. 5). The morphology of the Huh7 and C3A cells cultured in alginate microspheres was not affected by ITE, and viability measured by FDA/PI staining indicated that ITE (0 and 1 μM) was minimally toxic to the growth of cells in alginate microspheres.

To explore whether ITE was able to further improve the transcription of metabolism-associated genes in the Huh7 and C3A cells cultured in microspheres, we examined the transcription levels of CYP450-related genes. Our results revealed that ITE increased the mRNA expression levels of CYP450-related genes, such as 1A2, 2B6, 2D6, UGT1A1, SUL1A2 and nuclear receptor Hecbpa with a >2-fold increase compared with the control group in the Huh7 microspheres, and 1A2, UGT1A1, COMT and hHNF-4a in the C3A microspheres (Fig. 6).

The CYP450 protein levels were then measured in the Huh7 and C3A cells cultured in microspheres. The CYP1A2, CYP3A4, CYP2E1, CYP1A1 and CYP1B1 proteins were detected by western blot analysis, which confirmed that ITE increased the protein levels of CYP450 enzymes in both the Huh7 and C3A cells (Fig. 7). The CYP3A4 and CYP2E1 protein levels were increased by ITE treatment both in Huh7 microspheres and C3A microspheres. The CYP1A1 protein level in C3A microspheres was increased after ITE treatment. By contrast, in Huh7 microspheres, the CYP1A1 protein level was increased only with 0.5 μM ITE, but decreased with 1 and 2 μM ITE. However CYP1A2 and CYP1B1 were not affected by ITE, which was inconsistent with their metabolic activities (shown below). Again, amongst the examined CYP450s, the greatest increase was observed in the protein levels of CYP3A4.

We evaluated the metabolic functions and synthesis capacity of the Huh7 and C3A cells cultured in alginate microspheres in the presence of ITE (Fig. 8). As already demonstrated in Huh7 and C3A cells on monolayer cultures, the metabolic activities of the main CYP450s (CYP1A1, CYP1A2, CYP1B1 and CYP3A4) in the Huh7 and C3A cells cultured in alginate microspheres significantly increased with ITE treatment (Fig. 8A–D and G–J). In the C3A microspheres, albumin secretion and urea synthesis were not affected by ITE (Fig. 8K and L). However, albumin secretion and urea synthesis in the Huh7 microspheres seemed to be slightly affected. In contrast to 0.5 μM ITE treatment which slightly increased the albumin secretion (Fig. 8E) and urea synthesis (Fig. 8F), higher concentrations (1 and 2 μM) of ITE decreased both albumin secretion (Fig. 8E) and urea synthesis (Fig. 8F). All these results indicated that ITE treatment led to significantly higher detoxification capacities of the liver cells, but had a lesser effect on their biological synthesis capabilities.

In order to explore the transcriptional profile of ITE-treated Huh7 and C3A cells on monolayer cultures, we performed a DNA microarray. We found that the ITE-treated C3A cells elicited quite different transcriptional profiles compared to the untreated cells (Fig. 9). By contrast, 0.2 μM ITE markedly altered processes involved in cell cycle, DNA replication and the metabolism of xenobiotics via the CYP450 system (Fig. 10). Moreover GO enrichment analysis further verified that processes, such as mitotic cell cycle, mitotic nuclear division and DNA replication were particularly altered by ITE treatment (Fig. 11), indicating a potential link between P450-mediated metabolism activities and cell cycle regulation.