Regulation of Biotransformation Systems and ABC Transporters by Benznidazole in HepG2 Cells: Involvement of Pregnane X-Receptor

Abstract

BackgroundBenznidazole (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 FindingsExpression 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/SignificanceModifications 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.

The trypanocidal benznidazole promotes adaptive response to oxidative injury: Involvement of the nuclear factor-erythroid 2-related factor-2 (Nrf2) and multidrug resistance associated protein 2 (MRP2)

“…In the absence of an agonist, PXR is predominantly associated to corepressors. Upon agonist binding, for example BZL (Rigalli et al 2012), PXR dissociates from its corepressors and binds to coactivators, which consequently leads to transcriptional activation of its target genes (Hariparsad et al 2009). Additionally, the nuclear erythroid 2-related factor 2 (Nrf2) is a transcription factor that plays a key role in orchestrating adaptive responses to oxidative stress.…”

Modulation of Biotransformation Systems and ABC Transporters by Benznidazole in Rats

“…Lower levels of PXR have also been detected in kidney (46). The knockdown of PXR in HepG2 cells was able to abolish the induction of CYP3A4, GST-, P-gp, and MRP2 by BZL, suggesting that this nuclear receptor is involved in BZL effects (12). We postulate that the differential induction of studied systems in liver and intestine versus those in kidney could be related to tissue-specific differences in PXR expression and/or other transcription factors.…”

“…The decreased mucosa to serosa transport of BZL in intestinal sacs confirms this assumption. The participation of P-gp in BZL efflux was observed in P-gp knockdown HepG2 cells (12). Further experiments are needed to corroborate the contribution of this transporter or any other in BZL transport in an in vivo model.…”

“…*, significantly different from control; (P Ͻ 0.05). ment in their activities in cells from a hepatoma cell line, HepG2, treated with BZL (200 M, 48 h) (12). Here we studied the effects of BZL treatment on phase I and phase II biotransformation enzymes and ABC efflux transporters in rat liver, small intestine, and kidney and the consequences in BZL disposition.…”

“…Recently we observed that BZL induces the expression and activities of CYP3A4, GST-, P-gp, and MRP2 in a concentrationdependent manner in HepG2 cells, used as model of human hepatocytes (12). However, at present there is no information concerning the expression and activities of biotransformation systems and transporters in an in vivo model and the potential consequences in pharmacokinetics of BZL or coadministered drugs.…”