Augmentation of 3-methylcholanthrene-induced bioactivation in the human hepatoma cell line HepG2 by the calcium channel blocker nicardipine

Abstract

The abilities of the dihydropyridine calcium channel blocker nicardipine (Nic) to induce cytochrome P450 1 family enzymes (CYP1s) and to enhance the 3-methylcholanthrene (MC)-mediated induction of CYP1s and formation of MC-DNA adduct were examined in the human hepatoma cell line HepG2. The results from real time RT-PCR analysis demonstrated that Nic could induce CYP1 mRNAs and enhance the MC-mediated induction of the CYP1 mRNAs. The luciferase-reporter gene assay using the HepG2-A10 cell line, which has been previously established for the screening of aryl hydrocarbon receptor (AhR) activators, also indicated the augmentation of MC-mediated activation of AhR (induction of luciferase) by Nic, although Nic showed limited capacity for the activation of AhR. Furthermore, the results from the Western blot analysis of CYP1s, the enzyme activity assay, and the assay for MC-DNA adduct formation indicated that Nic could enhance the MC-mediated induction of CYP1s, especially CYP1A1. Furthermore, the intracellular accumulation level of [(3)H]MC after treatment of HepG2 cells with [(3)H]MC significantly increased in the presence of Nic. The present findings demonstrate that Nic can enhance the MC-mediated induction of CYP1s, especially CYP1A1, and the formation of MC-DNA adduct in HepG2 cells. Furthermore, the augmentation of the MC-mediated bioactivation by Nic is demonstrated to occur mainly through an increase in intracellular accumulation of MC.

Figure 1

Temporal changes in the mRNA levels of the cytochrome P450 1 family enzymes (CYP1s), after treatment of HepG2 cells with nicardipine (Nic), 3‐methylcholanthrene (MC), or their combination. HepG2 cells were treated with 0.2% DMSO (Cont), 10 μM Nic, 30 nM MC, or a combination of Nic and MC for the indicated times. The mRNA levels were analyzed by real time RT‐PCR. Expression levels of the mRNAs of (a) CYP1A1, (b) CYP1A2, and (c) CYP1B1 were normalized to that of GAPDH mRNA and are shown as the ratio to time 0. The symbols and bars represent the means and their SDs, respectively (n = 3). Significant differences from the time‐matched controls, *P < 0.05, **P < 0.01. (d) Representative expression patterns of CYP1A1, CYP1A2, and CYP1B1 mRNAs. Equal volumes of the PCR mixtures were subjected to electrophoresis on a 2% agarose gel, as previously described.^{( 12 )} The values in parentheses represent the numbers of PCR cycles performed.

Figure 2

Temporal changes in xenobiotic response element‐driven luciferase activity after treatment of HepG2‐A10 cells with nicardipine (Nic), 3‐methylcholanthrene (MC), or their combination. HepG2‐A10 cells were treated with 0.2% DMSO (Cont), 10 μM Nic, 30 nM MC, or a combination of Nic and MC for the indicated times. The luciferase activity was measured as described in the Materials and Methods, and is shown as the ratio to time 0. The symbols and bars represent the means and their SDs, respectively (n = 4). Significant differences from the time‐matched controls, *P < 0.01.

Figure 3

Changes in ethoxyresorufin O‐deethylation (EROD) activity after treatment of HepG2 cells with nicardipine (Nic), 3‐methylcholanthrene (MC), or their combination. (a) Temporal changes in EROD activity after chemical treatments. HepG2 cells were treated with 0.2% DMSO (Cont), 10 μM Nic, 30 nM MC, or a combination of Nic and MC for the indicated times. The symbols and bars represent the means and their SDs, respectively (n = 4). Significant differences from the time‐matched controls, *P < 0.01. (b) Dose effect of Nic on the MC‐mediated induction of EROD activity. HepG2 cells were treated for 24 h with Nic (0.1, 0.3, 1, 3, or 10 μM), MC (30 nM), or a combination of Nic and MC. The values and bars represent the means and their SDs, respectively (n = 4). Significant differences between chemical‐untreated (control) and Nic‐treated HepG2 cells, *P < 0.01. Significant differences between the HepG2 cells treated with MC alone and a combination of MC and Nic, #P < 0.01.

Figure 4

Representative expression patterns of cytochrome P450 1A (CYP1A) subfamily enzyme proteins in HepG2 cells treated with nicardipine (Nic), 3‐methylcholanthrene (MC), or their combination. HepG2 cells were treated with 0.2% DMSO (Cont), 10 μM Nic, 30 nM MC, or a combination of Nic and MC for 24 h. A portion (20 μg protein/lane) of the S‐12 fraction prepared from the cell lysate was used for Western blot analysis, as described in the Materials and Methods. CYP1A2 protein was not detected in the cells treated with any chemicals. Similar results were obtained in three other experiments.

Figure 5

Effect of nicardipine (Nic) on the formation of the 3‐methylcholanthrene (MC)‐DNA adduct in HepG2 cells. HepG2 cells were treated with 30 nM [³H]MC in the presence or absence of 10 μM Nic for 24 h. Total genomic DNAs were prepared from the cells, and the amounts of [³H]MC covalently bound to DNA were determined by liquid scintillation counting, as described in the Materials and Methods. Each column indicates the mean in each treatment group, and bars represent the SDs (n = 4). Significant differences from the cells treated with MC alone were assessed by the Student’s t‐test; *P < 0.01.

Figure 6

Effect of nicardipine (Nic) on the intracellular accumulation of 3‐methylcholanthrene (MC) in HepG2 cells. HepG2 cells were treated with 30 nM [³H]MC in the presence or absence of 10 μM Nic for the indicated times, and the intracellular accumulation levels of [³H]MC were measured by liquid scintillation counting, as described in the Materials and Methods. The symbols and bars represent the means and their SDs, respectively (n = 4). Significant differences from the time‐matched cells treated with MC alone were assessed by the Student’s t‐test; *P < 0.01.