Regulation of biotransformation systems and ABC transporters by benznidazole in HepG2 cells: involvement of pregnane X-receptor

Abstract

Background: Benznidazole (BZL) is the only antichagasic drug available in most endemic countries. Its effect on the expression and activity of drug-metabolizing and transporter proteins has not been studied yet.

Methodology/principal findings: Expression and activity of P-glycoprotein (P-gp), Multidrug resistance-associated protein 2 (MRP2), Cytochrome P450 3A4 (CYP3A4), and Glutathione S-transferase (GST) were evaluated in HepG2 cells after treatment with BZL. Expression was estimated by immunoblotting and real time PCR. P-gp and MRP2 activities were estimated using model substrates rhodamine 123 and dinitrophenyl-S-glutathione (DNP-SG), respectively. CYP3A4 and GST activities were evaluated through their abilities to convert proluciferin into luciferin and 1-chloro-2,4-dinitrobenzene into DNP-SG, respectively. BZL (200 µM) increased the expression (protein and mRNA) of P-gp, MRP2, CYP3A4, and GSTπ class. A concomitant enhancement of activity was observed for all these proteins, except for CYP3A4, which exhibited a decreased activity. To elucidate if pregnane X receptor (PXR) mediates BZL response, its expression was knocked down with a specific siRNA. In this condition, the effect of BZL on P-gp, MRP2, CYP3A4, and GSTπ protein up-regulation was completely abolished. Consistent with this, BZL was able to activate PXR, as detected by reporter gene assay. Additional studies, using transporter inhibitors and P-gp-knock down cells, demonstrated that P-gp is involved in BZL extrusion. Pre-treatment of HepG2 cells with BZL increased its own efflux, as a consequence of P-gp up-regulation.

Conclusions/significance: Modifications in the activity of biotransformation and transport systems by BZL may alter the pharmacokinetics and efficiency of drugs that are substrates of these systems, including BZL itself.

Figure 1. Effect of BZL on transporter expression in cellular lysates.

P-gp (panel A) and MRP2 (panel B) were detected by western blotting in HepG2 total lysates after 48 h of treatment with BZL (2, 20, 200 and 1000 µM) or vehicle (C). Equal amounts of total protein (15 µg) were loaded in the gels. MRP2 and P-gp O.D. were related to β-actin O.D. Uniformity of loading and transfer from gel to PVDF membrane was also controlled with Ponceau S. The data on O.D. (% of C) are presented as mean ± S.D. (n = 3). Typical western blot detections are shown at the bottom. *Significantly different from C, p<0.05; **Significantly different from C, p<0.01; ***Significantly different from C, p<0.001.

Figure 2. Effect of BZL on transporter expression in crude plasma membranes.

P-gp (panel A) and MRP2 (panel B) were detected by western blotting in HepG2 plasma membranes after 48 h of treatment with BZL (200 µM) or vehicle (C). Equal amounts of total protein (5 µg) were loaded in the gels. MRP2 and P-gp O.D. were related to β-actin O.D. Uniformity of loading and transfer from gel to PVDF membrane was also controlled with Ponceau S. The data on O.D. (% of C) are presented as mean ± S.D. (n = 3). Typical western blot detections are shown at the bottom. *Significantly different from C, p<0.05.

Figure 3. Effect of BZL on P-gp activity.

Accumulation of Rh123, in the presence or absence of verapamil (VER; 100 µM), was inversely correlated with P-gp activity in cells pretreated with BZL (200 µM) or vehicle (C) for 48 h. Data are presented as percentages referred to the accumulation in C, considered as 100%, and were expressed as means ± S.D. (n = 3). a: significantly different from C; b: significantly different from C+VER; c: significantly different from BZL. Significance levels were set at p<0.05.

Figure 4. Effect of BZL on MRP2 activity.

Extrusion of DNP-SG in the presence or absence of probenecid (PRO; 1 mM), was determined in supernatants of cells pretreated with BZL (200 µM, 48 h) or vehicle (C) by HPLC. Samples were taken after 60 min of incubation. Data (means ± S.D, n = 3) are presented as percentage of DNP-SG extruded in control cells. a: significantly different from C; b: significantly different from C+PRO; c: significantly different from BZL. Significance levels were set at p<0.05.

Figure 5. Effect of BZL on CYP3A4 and GST expression.

Cells were exposed either to vehicle (C) or BZL (200 µM) for 48 h. CYP3A4 (panel A), GSTα (panel B), GSTμ (panel C), and GSTπ (panel D) levels were estimated by western blotting. Equal amounts of total protein (15 µg) were loaded in the gels. CYP3A4 or GST O.D. was related to β-actin O.D. Uniformity of loading and transfer from gel to PVDF membrane was also controlled with Ponceau S. The data on O.D. (% of C) are presented as mean ± S.D. (n = 3). Typical western blot detections are shown at the bottom. *Significantly different from C, p<0.05.

Figure 6. Role of P-gp in BZL transport.

A. Confluent HepG2 cells were loaded with BZL (100 µM, 2 h) in the presence of either verapamil (VER; 100 µM) or probenecid (PRO, 1 mM). Control cells (C) were exposed to inhibitors vehicle. BZL accumulation was determined in cellular lysates by HPLC. Data (means ± S.D, n = 3) are expressed as percentage of BZL accumulated in control cells. *Significantly different from all the other groups, p<0.05. B. P-gp levels were estimated by western blotting in lysates from HepG2 cells transfected either with 100 nM Control siRNA-A (P-gp⁺) or with 100 nM Mdr-1 (h) si-RNA (P-gp⁻). Equal amounts of total protein (7 µg) were loaded in the gels. O.D. from P-gp was related to GAPDH O.D. Typical western blot detections from both groups are shown at the bottom. The results (% of P-gp⁺ cells) are expressed as mean ± S.D. (n = 3). *Significantly different from P-gp⁺, p<0.05. C. P-gp⁺ and P-gp⁻ cells were loaded with BZL (100 µM, 2 h). BZL accumulation was determined in cellular lysates by HPLC. Data (means ± S.D., n = 4) are expressed as percentage of BZL accumulated in P-gp⁺ cells. *Significantly different from P-gp⁺.

Figure 7. Effect of BZL on its own transport.

HepG2 cells were pretreated with BZL in conditions shown to induce P-gp expression (200 µM, 48 h) or (C) vehicle. Then they were loaded with BZL (100 µM, 2 h) with or without verapamil (VER; 100 µM). BZL accumulation was determined in cellular lysates by HPLC. Data (means ± S.D, n = 3) are expressed as percentage of BZL accumulated in control (C) cells. a: significantly different from C; b: significantly different from C+VER; c: significantly different from BZL, p<0,05.

Figure 8. Effect of PXR knock down on BZL mediated P-gp, MRP2, CYP3A4 and GSTπ induction.

P-gp (panel A), MRP2 (panel B), CYP3A4 (panel C) and GSTπ (panel D) levels were estimated by western blotting in lysates from HepG2 cells transfected either with 100 nM Control siRNA-A (PXR⁺) or 100 nM PXR siRNA (h) (PXR⁻) and exposed to BZL (200 µM, 48 h) or vehicle (C). Equal amounts of total protein (7 µg) were loaded in the gels. O.D. from each protein was related to GAPDH O.D. Uniformity of loading and transfer from gel to PVDF membrane was also controlled with Ponceau S. Typical western blot detections from each group are shown at the bottom of bar graphics. The results (% of each control) are expressed as mean ± S.D. (n = 3). *Significantly different from C, p<0.05.

Figure 9. BZL-mediated activation of PXR.

PXR activation was measured through the activation of the firefly luciferase gene under control of two PXR responsive elements after treatment with different concentrations of BZL (panel A) or RIF as positive control (panel B).