Nrf2 protects human bladder urothelial cells from arsenite and monomethylarsonous acid toxicity

Abstract

Arsenic is widely spread in our living environment and imposes a big challenge on human health worldwide. Arsenic damages biological systems through multiple mechanisms including the generation of reactive oxygen species. The transcription factor Nrf2 regulates the cellular antioxidant response that protects cells from various insults. In this study, the protective role of Nrf2 in arsenic toxicity was investigated in a human bladder urothelial cell line, UROtsa. Using a UROtsa cell line stably infected with Nrf2-siRNA, we clearly demonstrate that compromised Nrf2 expression sensitized the cells to As(III)- and MMA(III)-induced toxicity. On the other hand, the activation of the Nrf2 pathway by tert-butylhydroquinone (tBHQ) and sulforaphane (SF), the known Nrf2-inducers, rendered UROtsa cells more resistant to As(III) and MMA(III). Furthermore, the wild-type mouse embryo fibroblast (WT-MEF) cells were protected from As(III)- and MMA(III)-induced toxicity following Nrf2 activation by tBHQ or SF, whereas neither tBHQ nor SF conferred protection in the Nrf2(-/-)MEF cells, demonstrating that tBHQ- or SF-mediated protection against As(III)- and MMA(III)-induced toxicity depends on Nrf2 activation. These results, obtained by both loss of function and gain of function analyses, clearly demonstrate the protective role of Nrf2 in arsenic-induced toxicity. The current work lays the groundwork for using Nrf2 activators for therapeutic and dietary interventions against adverse effects of arsenic.

Fig. 1. UROtsa-725 had reduced endogenous Nrf2

(A) UROtsa-725 and its negative control URotsa-724 were derived from URotsa cells stably infected with Nrf2-siRNA or scrambled-siRNA using retrovirus-based vectors. Equal amounts of total lysates were subjected to immunoblot analysis with anti-Nrf2 and anti-tubulin antibodies. (B) The mRNA levels of Nrf2, Keap1, NQO1, and HO-1 in UROtsa-724 and UROtsa-725 were compared by real-time RT-PCR. (C) NQO1 activities in the lysates of UROtsa-724 and UROtsa-725 were measured using DCPIP as a substrate. (D) Intracellular glutathione concentrations in UROtsa-724 and UROtsa-725 were measured using the QuantiChrom glutathione assay kit. (E) UROtsa-724 and UROtsa-725 cell lines were either left untreated or treated with 20 μM As(III) for 16 h. Following incubation of cells with DCF, ROS was detected using flow cytometry. The bar graph represents the means of fluorescence values.

Fig. 2. Down-regulation of Nrf2 sensitizes cells to As(III)- and MMA(III)-induced toxicity

UROtsa-724 and UROtsa-725 were treated with the indicated concentrations of As(III) (A) or MMA(III) (B) for 24h. Cell viability was measured by the MTT assay.

Fig. 3. tBHQ or SF pretreatment protects cells from As(III)- and MMA(III)-induced toxicity

(A and B) UROtsa cells were pretreated and co-treated with DMSO (diamond), tBHQ (triangle) or SF (square). Treatment conditions are: 40 μM (24 h) pretreatment and 5 μM (24h) cotreatment for tBHQ, 8 μM (24 h) pretreatment and 0.5 μM (24h) cotreatment for SF. During the cotreatment time period, 5 μM tBHQ or 0.5 μM SF was added along with the indicated concentrations of As(III) or MMA(III). (C–F) Cell viability assay was carried out in WT-MEF (C and D) and Nrf2^−/− MEF cells (E and F). The pretreatment and cotreatment conditions for tBHQ or SF were same as described in Figure 3A or 3B.