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)

Abstract

Oxidative stress is a frequent cause underlying drug-induced hepatotoxicity. Benznidazole (BZL) is the only trypanocidal agent available for treatment of Chagas disease in endemic areas. Its use is associated with side effects, including increases in biomarkers of hepatotoxicity. However, BZL potential to cause oxidative stress has been poorly investigated. Here, we evaluated the effect of a pharmacologically relevant BZL concentration (200μM) at different time points on redox status and the counteracting mechanisms in the human hepatic cell line HepG2. BZL increased reactive oxygen species (ROS) after 1 and 3h of exposure, returning to normality at 24h. Additionally, BZL increased glutathione peroxidase activity at 12h and the oxidized glutathione/total glutathione (GSSG/GSSG+GSH) ratio that reached a peak at 24h. Thus, an enhanced detoxification of peroxide and GSSG formation could account for ROS normalization. GSSG/GSSG+GSH returned to control values at 48h. Expression of the multidrug resistance-associated protein 2 (MRP2) and GSSG efflux via MRP2 were induced by BZL at 24 and 48h, explaining normalization of GSSG/GSSG+GSH. BZL activated the nuclear erythroid 2-related factor 2 (Nrf2), already shown to modulate MRP2 expression in response to oxidative stress. Nrf2 participation was confirmed using Nrf2-knockout mice in which MRP2 mRNA expression was not affected by BZL. In summary, we demonstrated a ROS increase by BZL in HepG2 cells and a glutathione peroxidase- and MRP2 driven counteracting mechanism, being Nrf2 a key modulator of this response. Our results could explain hepatic alterations associated with BZL therapy.

Keywords: Benznidazole; Multidrug resistance associated protein 2; Nuclear factor-erythroid 2-related factor-2; Oxidative stress.

Fig. 1. Redox status in BZL treated HepG2 cells

Indicators of intracellular redox status were determined in control and BZL-treated cells (200 μM; 15 min, 1 h, 3 h, 24 h and 48 h). tBOOH (500 μM, 15 min) was used as positive control of ROS generation. a. intracellular ROS, b. GSSG/GSSG+GSH ratio was calculated measuring from the intracellular GSSG and total glutathione (GSSG+GSH), c. Intracellular GSSG content. d. Total intracellular glutathione (GSSG+GSH). e. Concentration-dependence of BZL effects on intracellular redox status. GSSG/GSSG+GSH ratio was assessed after incubation with 50, 100 and 200 μM BZL (24 h). All measurements (mean ± S.D., n = 3-4) are expressed as percentage of control values (C). * different from C, p<0.05.

Fig. 2. MRP2 expression and MRP2 mediated GSSG efflux in BZL treated HepG2 cells

a. MRP2 expression in lysates from BZL treated cells (200 μM, 24 and 48 h). Fifteen μg of total protein were loaded in the gels. MRP2 O.D. was normalized to β-actin O.D. Uniformity of loading and transfer from gel to PVDF membrane was also determined by Ponceau S staining. Results (mean ± S.D., n = 3) are expressed as percentage of control values (C). * different from C, p<0.05. b. GSSG efflux in BZL treated cells (200 μM, 24 and 48 h). GSSG was determined in culture medium of HepG2 cells after 24 and 48 h of treatment with BZL. MK571 (10 μM) was used as MRP2 inhibitor. Experiments were performed in absence (-MK571) and presence (+MK571) of MK571. tBOOH (500 μM, 15 min) was used as a model of rapid ROS generation with increase in intracellular GSSG without MRP2 induction. Results (mean ± S.D., n = 3) are expressed as percentage of control values (C) from cells without MK571. a: different from C-MK571, b: different from BZL 24 h-MK571, c: different from BZL 48 h-MK571, d: different from tBOOH-MK571, p<0.05.

Fig. 3. PXR participation in MRP2 induction by BZL in HepG2 cells

HepG2 cells were transfected with a control non-silencing- (PXR⁺ cells) or with a PXR specific siRNA (PXR⁻ cells). a. PXR protein levels in cell lysates of PXR⁺ and PXR⁻ cells were assessed through western blot. PXR O.D. was normalized to GAPDH O.D. Results (mean ± S.D., n = 3) are expressed as percentage of the ratio in PXR⁺ cells. * different from PXR⁺, p<0.05. b. To further verify PXR knock-down, CYP3A4 expression was assessed through western blot in PXR⁺ and PXR⁻ HepG2 cells treated with RIF (20 μM, 24 h) or vehicle (C). CYP3A4 O.D. in control and RIF treated cells was normalized to GAPDH O.D. Results (mean ± S.D., n = 3) are expressed as percentage of the corresponding control. * different from control, p<0.05. c. MRP2 expression was quantified in total lysates of PXR⁺ and PXR⁻ cells exposed to BZL (200 μM, 24 h) or vehicle (C). MRP2 O.D. was normalized to GAPDH O.D. Results (mean ± S.D., n = 3) are expressed as percentage of the ratio in PXR⁺ cells. * different from the corresponding control, p<0.05.

Fig. 4. Participation of Nrf2 in MRP2 modulation by BZL in HepG2 cells

Purity of nuclear and cytosolic fractions was checked assessing H1 Histone and GAPDH enrichment, respectively (a). Nrf2 expression was quantified in nuclear extracts (b) and cytosolic fraction (c) of BZL treated HepG2 cells (200 μM; 3, 24 and 48 h). Equal amounts of protein were loaded in the gels. Nrf2 O.D. was normalized to histone H1 and GAPDH O.D. in nuclear and cytosolic fractions, respectively. Uniformity of loading and transfer from gel to PVDF membrane was also determined by Ponceau S staining. Firefly luciferase activity was assessed in HepG2 cells transfected with pGL3-ARE and exposed to BZL (200 μM; 3, 24 and 48 h) (d). Results (mean ± S.D., n = 3-4) are expressed as percentage of control values (C). * different from C, p<0.05. For a final confirmation of Nrf2 participation, an Nrf2 knock-down model was established. e: Nrf2 expression was assessed in total lysates of Nrf2⁺ (transfected with a non-silencing siRNA) and Nrf2⁻ HepG2 cells (transfected with a pool of specific siRNAs against human Nrf2) and normalized to GAPDH expression. Results (mean ± S.D., n = 3) are expressed as percentage of the Nrf2/GAPDH ratio in Nrf2⁺ cells. * different from Nrf2⁺, p<0.05. f: MRP2 expression was assessed in control and BZL-treated (200 μM, 24 h) Nrf2⁺ and Nrf2⁻ cells. Results (mean ± S.D., n = 3) are expressed as percentage of the Nrf2/GAPDH ratio in the corresponding control cells. * different from control (C), p<0.05.

Fig. 5. Effect of BZL on hepatic Mrp2 expression in wild type and Nrf2^−/− mice

Hepatic Mrp2 expression was quantified at the mRNA level in wild type and Nrf2 knock out control (vehicle exposed) and BZL treated mice (200 mg/kg b.w./ day, 3 consecutive days, i.p.) by real time PCR. Expression of Mrp2 (Abcc2) mRNA was normalized to β-actin mRNA. The data (mean ± S.D., n = 3) are expressed as percentage of the mRNA in the corresponding control (C) mice of each genotype. * different from the corresponding C, p<0.05.