Introduction

Groundwater arsenic contamination poses a dreadful threat to millions of people across the world. More than 10 million residents in Southeast Asia are estimated to be at risk from consuming arsenic-contaminated groundwater. Arsenic has been detected from several parts of Bangladesh, India, China, Nepal, Pakistan, and most Southeast Asian countries including Cambodia, Vietnam, Myanmar, Lao People’s Democratic Republic, Thailand, Taiwan, and Indonesia (Kim et al. 2011). Many states within the United States have been reported with significant concentrations (up to 50 ppm) of arsenic in the groundwater (Tchounwou et al. 2003; Knobeloch et al. 2006). In Latin America, the problem of arsenic contamination in water is known in 14 out of 20 countries: Argentina, Bolivia, Brazil, Chile, Colombia, Cuba, Ecuador, El Salvador, Guatemala, Honduras, Mexico, Nicaragua, Peru, and Uruguay (Bundschuh et al. 2011). In countries such as Romania, Hungary, Italy, and Spain, elevated arsenic concentrations have been detected, and special treatment steps have been recommended to reduce the arsenic to acceptable levels (van Halem et al. 2009). However, a number of large aquifers with arsenic naturally occurring at concentrations greater than World Health Organization (WHO) permissible limit of 10 μg/L (WHO 1993) or even significantly higher have been identified in several parts of the world (Kim et al. 2011). Some of the worst reports have been evidenced from Bangladesh and West Bengal in India. Water analyses of all 64 districts of Bangladesh reported 27.2 and 42.1 % of the tubewells with arsenic above 50 and 10 μg/L, respectively, and 7.5 % contained arsenic above 300 μg/L (Chakraborti et al. 2010). In all the 19 districts of West Bengal, India, 48.1 % have arsenic above 10 μg/L (WHO guideline), 23.8 % above 50 μg/L (Indian standard), and 3.3 % above 300 μg/L (concentration expected to produce overt arsenical skin lesions) (Chakrabarti et al. 2009).

Neoplastic and non-neoplastic clinical manifestations of arsenic

Inorganic arsenic is classified by the International Agency for Research on Cancer (IARC 2011), the Agency for Toxic Substances and Disease Registry (ATSDR 2012), and the Integrated Risk Information System (IRIS 2011) of the United States Environmental Protection Agency as a known human carcinogen. This classification was based on several epidemiological studies, which have shown a positive association between arsenic exposure and development of cancer in several countries (Kapaj et al. 2006). Chronic exposure to arsenic can lead to lung, bladder, liver, kidney, colon, prostate, and skin cancers (Kim et al. 2011). Skin cancer is the most common form of malignancy related to arsenic ingestion. Lung cancer is the most deadly form of cancer associated with arsenic exposure (Smith et al. 1992; Mead 2005). For internal organs, bladder cancer exhibits the highest relative risk. A small but measurable increase in the incidence of bladder cancer was associated with exposure to concentration as low as 10 ppm of inorganic arsenic (Chu and Crawford-Brown 2006). Other types of cancer associated with arsenic exposure include kidney, liver, and prostate cancers (Chen et al. 1992; Bates et al. 1992). Both neoplastic and non-neoplastic effects have been described as a consequence of arsenic exposure. Non-neoplastic effects primarily include peripheral vascular disorders (e.g., “black foot disease” almost exclusively observed in zones of Taiwan affected by arsenic contamination), hypertension, diabetes, severe atherosclerosis, neuropathies, and importantly skin alterations, such as hyperkeratosis and hyperpigmentation. Hyperkeratosis is particularly relevant, because it has been described as a precursor lesion of skin cancer tumors associated with arsenic exposure. Recent studies indicate that prenatal arsenic exposure also increases the risk of adverse effects during early childhood. The modes of action included epigenetic effects, mainly via DNA hypomethylation, endocrine effects (most classes of steroid hormones), immune suppression, neurotoxicity, and interaction with enzymes critical for fetal development and programming (Vahter 2008).

Multifactorial determinants are interwoven with mode of action of arsenic

The arsenicals in drinking water are mainly in inorganic pentavalent and trivalent forms. In the human body, pentavalent arsenate (AsV) is reduced to trivalent arsenite (AsIII) and then methylated to monomethylarsonic acid (MMAV), monomethylarsonous acid (MMAIII), dimethylarsinic acid (DMAV), and dimethylarsinous acid (DMAIII) by the alternate reduction of arsenic from pentavalent to trivalent forms and the addition of methyl groups (Rossman 2003). Earlier the methylation pathway was considered to be a method of detoxification, but later studies indicated that MMAIII and DMAIII are more toxic than inorganic trivalent arsenic (Mass et al. 2001; Chen et al. 2003). As V, As III, MMAIII, MMAV, DMAIII, and DMAV are all detectable in the urine of people who drink arsenic-contaminated water (Mandal et al. 2004). Arsenic alters multiple cellular pathways including expression of growth factors, suppression of cell cycle checkpoint proteins, promotion of and resistance to apoptosis, inhibition of DNA repair, alterations in DNA methylation, decreased immunosurveillance, and increased oxidative stress, by disturbing the pro/antioxidant balance. These alterations play prominent roles in disease manifestation, such as carcinogenicity, genotoxicity, diabetes, and cardiovascular and nervous systems disorders (Flora 2011). The mode of action of arsenicals is quite complicated, and the multifactorial determinants range from the valence state (trivalent/ pentavalent), degree of methylation, charge at physiological pH, and electrostatic attraction and repulsion to active sites on important macromolecules, to pharmacokinetic factors (absorption, distribution, metabolism, and excretion).

Oxidative stress plays a cardinal role in arsenic poisoning

Oxidative stress is the most widely accepted and studied mechanism of arsenic toxicity (Ercal et al. 2001). Oxidative stress represents an imbalance between the production and manifestation of reactive oxygen species (ROS) and a biological system’s ability to readily detoxify the reactive intermediates or to repair the resulting damage. Oxidative stress is basically caused by two main mechanisms. The concentration of antioxidants is reduced due to mutated antioxidant enzymes, toxins or the reduced intake of natural antioxidants (Gülçin 2012). Review of literature suggested that ROS and reactive nitrogen species (RNS) are involved in arsenic toxicity (Bau et al. 2002; Gurr et al. 2003; Shi et al. 2004). Major arsenic-induced ROS include superoxide anion (O ·−2 ), hydroxyl radical (·OH), hydrogen peroxide (H2O2), singlet oxygen (1O2), and peroxyl radicals (Flora 2011). Oxygen-derived radicals form a most important class of radical species generated in living systems due to unique electronic configuration of molecular oxygen that forms O ·−2 by addition of one electron (Miller et al. 1990). Molecular oxygen reacts with dimethylarsine (a trivalent arsenic form and a minor in vivo metabolite of DMAV) to form dimethylarsinic radical and superoxide anion. Further, the addition of another molecule of molecular oxygen results in a dimethylarsinic peroxyl radical, and these arsenic radicals are known to be detrimental to cells (Yamanaka et al. 1990). ROS also caused a number of non-enzymatic modifications to proteins, including carbonylation, o-tyrosine, chlorotyrosine, nitrotyrosine, and dityrosine (Reilly and Aust 1999). Arsenic induced morphologic changes in mitochondrial integrity and a rapid decline of mitochondrial membrane potential. Mitochondrial alterations are considered to be primary sites where an uncontrolled random formation of O ·−2 occurs. Cascade of free radical formation derived from the O ·−2 , combined with glutathione (GSH)-depleting agents, increased the sensitivity of cells to arsenic toxicity (Jomova et al. 2011). Chronic arsenic exposure induced oxidative stress by causing DNA damage, increasing lipid peroxidation and protein carbonyl formation, and depleting antioxidants in human lymphocytes (Biswas et al. 2010a). Arsenic disturbed the redox homeostasis and induced oxidative DNA adducts in Swiss albino mice (Sinha et al. 2010; Sinha and Roy 2011).

Nuclear factor-erythroid-2 p45-related factor (Nrf2) as a redox-sensitive transcription factor

Nrf2 is a redox-sensitive transcription factor that positively regulates the expression of genes encoding antioxidants, xenobiotic detoxification enzymes, and drug efflux pumps and confers cytoprotection against oxidative stress and xenobiotics in normal cells (Singh et al. 2006). Nrf2 is a basic-leucine zipper transcription factor that binds to the antioxidant response element (ARE) promoter sequence, leading to the coordinated upregulation of ARE-driven detoxifying and antioxidant genes. The Nrf2 downstream genes identified so far can be grouped into several categories, including (1) intracellular redox-balancing proteins, such as glutamate cysteine ligase (GCL), glutathione peroxidase, thioredoxin (Trx), thioredoxin reductase (TrxR), peroxiredoxin, and heme oxygenase-1 (HO-1); (2) phase II detoxifying enzymes such as glutathione S-transferase (GST), NAD(P)H quinone oxidoreductase-1 (NQO1), and UDP-glucuronosyltransferase (UGT); and (3) transporters such as multidrug resistance-associated protein (MRP) (Lau et al. 2008). Coordinated upregulation of the Nrf2-mediated endogenous antioxidants, phase II detoxifying enzymes, and drug transporters is essential in defending cells or animals from damage by environmental insults (Jiang et al. 2009). Nrf2 maintains intracellular redox balance through transcriptional upregulation of an array of downstream genes, such as GCL, HO-1, GST, MRPs, and NQO1 (Lau et al. 2008; Hayes and McMahon 2009). Nrf2 is a critical element in transactivating phase II enzyme expression through the cis-acting element, ARE (Hu et al. 2010). One or more AREs (A/G)TGA(C/T)nnnGC(A/G) are found in the 5′-flanking regions of many Nrf2 target genes, including the prototypic inducible genes GSTA1 and NQO1 (Kwak and Knesler 2010). Genomic analyses indicated that gene families affected by Nrf2 (a) provide direct antioxidants; (b) encode enzymes that directly inactivate oxidants; (c) increase the levels of GSH synthesis and regeneration; (d) stimulate NADPH synthesis; (e) enhance toxin export through the multidrug response transporters; (f) enhance the recognition, repair, and removal of damaged proteins; (g) elevate nucleotide excision repair; (h) regulate expression of other transcription factors, growth factors and receptors, and molecular chaperones; and (i) inhibit cytokine-mediated inflammation (Wakabayashi et al. 2010).

Regulation of Nrf2/ARE signaling pathway

Adaptation to oxidative stress created by ROS, RNS, and numerous electrophilic compounds is enabled by concerted upregulation of ARE-driven genes. There have been a number of hypotheses regarding the nuclear accumulation of Nrf2- and ARE-driven gene induction, which may confer unique functional operation under different physiological conditions.

Kelch-like ECH-associated protein 1 (Keap1)–Nrf2 regulatory complex

The main signaling pathway involved in the oxidative stress response is the Keap1–Nrf2–ARE pathway. Nrf2 signaling is the major cellular defense to relieve oxidative and electrophilic stress. Nrf2 induces antioxidant responsive genes, such as HO-1, catalase, and superoxide dismutase (SOD), and regulates phase II drug metabolism genes, including NQO1 (Kobayashi et al. 2006).

Regulation of Nrf2 under quiescent conditions

Keap1 is responsible for cytoplasmic–nuclear shuttling and proteasomal degradation of Nrf2 (Itoh et al. 2003; McMahon et al. 2003; Zhang and Hannink 2003). Under quiescent conditions, Nrf2 is anchored in the cytoplasm through binding to Keap1, which in turn facilitates the ubiquitylation and subsequent proteolysis of Nrf2 (Itoh et al. 1999; Tong et al. 2006). Intracellular existence of Keap1 is in the dimer form and comprises five distinct domains: (1) the N-terminal region (NTR, amino acids 1–60); (2) the BTB (Bric-a-brac, Tramtrack, Broad-complex) domain (amino acids 61–178), an evolutionarily conserved protein–protein interaction motif found in actin-binding proteins and zinc finger transcription factors, participates in the binding to Rbx1-bound Cul3 and the formation of homodimers; (3) intervening region (IVR, amino acids 179–321), a central linker domain, especially rich in cysteine, also takes part in binding to Rbx1-bound Cul3; (4) the Kelch-repeat domain (amino acids 322–608) mediates binding to the Neh2 domain of Nrf2; and (5) C-terminal region (CTR, amino acids 609–625) (Zhao et al. 2010). The Nrf2-ECH homology (Neh) domains of Nrf2 are highly conserved, and among them, the Neh2 domain mediates the binding of Nrf2 to Keap1, while other domains, such as Neh4 and Neh5, are known to mediate the transcriptional activity of Nrf2 (Itoh et al. 1999; Kobayashi et al. 2002; McMahon et al. 2004). Keap1 is a cysteine-rich protein, and some of the 27 cysteine residues in Keap1 are postulated to play a sensory role in detecting oxidants and xenobiotics (Motohashi and Yamamoto 2004). Certain specific cysteine residues like C257, C273, C288, and C297 have been reported to interact with the N-terminal Neh2 domain of Nrf2 (Kobayashi et al. 2002). Mutations of both C273 and C288 abolish the repressive effect of Keap1 on Nrf2, suggesting a critical role of these two cysteine residues in the repression of Nrf2 (Wakabayashi et al. 2004). Keap1 serves as an adaptor protein between Nrf2 and the Cullin3-based E3-ligase ubiquitylation complex, with its N-terminal BTB leading to ubiquitylation of Nrf2 and subsequent degradation by the 26S proteasome (Cullinan et al. 2004; Zhang et al. 2004). The central linker domains bind to Rbx1-bound Cul3 and its C-terminal Kelch domain binds to the Neh2 domain of Nrf2, thereby leading to ubiquitination and degradation of Nrf2. Recent studies have found two sites within the Neh2 domain of Nrf2, termed the LxxQDxDLG and DxETGE motifs, which mediate binding to the Keap1 Kelch repeats. The lysine residues within the Neh2 domain located at a distance of 10–30 amino acids on the N-terminal side of the DxETGE motif are targeted for ubiquitin transfer mediated by the Cul3-associated Rbx1 protein and an ubiquitin-charged E2 protein. Binding of Nrf2 to Keap1 via the DxETGE motif would position these residues for ubiquitin transfer and then facilitate the ubiquitination and degradation of Nrf2 (Zhao et al. 2010). Under normal conditions, the half-life of Nrf2 in mammalian cells is about 15–45 min, depending on cell type, and this turnover is mediated primarily by the ubiquitin-26S proteasome pathway (Jeong et al. 2006). Keap1, therefore, negatively regulates Nrf2 by both preventing its nuclear accumulation and enhancing its rate of proteasomal degradation (McMahon et al. 2003).

Regulation of Nrf2 activation

Cytoplasmic localization of Keap1 protein is known to be determined by both the Kelch/double glycine repeat domain, which mediates binding of Keap1 to actin cytoskeleton, and the IVR domain, which contains a nuclear export signal (Li and Kong 2009). A nuclear localization signal in the Nrf2 protein is known to facilitate its translocation from the cytoplasm into the nucleus (Jain et al. 2005; Li et al. 2006). Within the nucleus, Nrf2 protein dimerizes with small MAF proteins to bind to the ARE (Itoh et al. 1997). It has been suggested that Nrf2 can also form heterodimers with other bZIP transcription factors, such as ATF4 for binding to the ARE (Venugopal and Jaiswal 1998; He et al. 2001). Small MAF proteins are the major binding partners of Nrf2 for binding to the ARE and subsequent transactivation. Therefore, sequestration and further degradation of Nrf2 in the cytoplasm are the mechanisms provided for the repressive effects of Keap1 on Nrf2 function (Kwak and Knesler 2010). The increased stability and activation of Nrf2 by certain stimuli seem to come from their ability to repress the Keap1-dependent degradation mechanism. Exposure to a number of endogenous activators, such as ROS, RNS, and exogenous Nrf2 inducers, such as heavy metals and chemopreventive agents, the Nrf2–Keap1 complex disassociates thereby rescuing Nrf2 from proteasomal degradation and the half-life of Nrf2 increases significantly (Dinkova-Kostova et al. 2005). Upon stress stimulation, the nuclear protein prothymosin α binds the Kelch-repeat domain and liberates Nrf2, thus activating its target genes (Bellezza et al. 2010).

Two general mechanisms for Nrf2 nuclear accumulation in response to inducers have been proposed. The first is downregulation of Nrf2 ubiquitination, through modification of cysteine thiols of Keap1 or phosphorylation of Nrf2, or both, thereby disrupting the Keap1–Cul3 and Keap1–Nrf2 complexes and then leading to dissociation of Nrf2 from Keap1. The second mechanism involves alteration of the nuclear import/export of Nrf2 (Zhao et al. 2010). The release of Nrf2 from Keap1 and subsequent translocation from cytoplasm to nucleus is mediated by several effectors under redox stress like modification of cysteine residues in Keap1, protein kinase C (PKC)-mediated Nrf2 phosphorylation at Ser40, casein kinase 2 (CK2), extracellular signal-regulated kinase 2 (ERK2), ERK5, glycogen synthase kinase-3β (GSK-3β), c-Jun N-terminal kinase 1 (JNK1), PKR-like ER-localized eIF2α kinase (PERK), and phosphoinositide-3-kinase (PI3K) (Bellezza et al. 2010). Mitogen-activated protein kinases (MAPKs), protein kinase C (PKC), phosphatidylinositol 3-kinase (PI3K), and RNA-dependant protein kinase-like endoplasmic reticulum kinase (PERK) have been implicated in the process of phosphorylation and Nrf2 activation. The Nrf2 transactivation domain may be upregulated by ERK and JNK pathways through the coactivator CBP (Shen et al. 2004). PI3K is another kinase proposed to regulate the Nrf2/ARE pathway (Lee et al. 2001). ARE-mediated rGSTA2 induction in the rat hepatoma H4IIE cells by tBHQ was found to be dependent on PI3K signaling (Kang et al. 2001). PERK, a transmembrane protein kinase, has been shown to phosphorylate Nrf2, resulting in its dissociation from Keap1; such phosphorylation also inhibited the reassociation of Nrf2/Keap1 complexes in vitro (Cullinan et al. 2003).

Besides disruption of the Keap1–Nrf2–Cul3 ubiquitination complex, alteration of the nuclear import/export of Nrf2 is also involved in the mechanism of Nrf2 nuclear localization and accumulation in response to ARE inducers. Recent studies have showed that both phosphorylation and cysteine modification of Nrf2 could affect Nrf2 nuclear localization. GSK-3β phosphorylated Fyn, a Src kinase [at unknown threonine residue(s)], and caused nuclear localization of Fyn. This in turn probably phosphorylated Nrf2 tyrosine 568. This resulted in nuclear export of Nrf2, binding to Keap1, and degradation of Nrf2, thereby shutting down the signaling response (Jain and Jaiswal 2006). On the other hand, GSK-3β, downstream of PI3K, can be inactivated by Akt1 (Salazar et al. 2006). Therefore, it was speculated that ARE inducers that activated the PI3K pathway could inhibit Nrf2 phosphorylation by GSK-3β, decrease nuclear export of Nrf2, and lead to nuclear accumulation of Nrf2. Modification of Nrf2 cysteine residue C183 located in Neh5 may also shut down the nuclear export of Nrf2 allowing Nrf2 nuclear accumulation (Li et al. 2006).

Transcriptional regulation of Nrf2

Analyses of global transcriptional responses indicated toward interactions between Nrf2 signaling and other prominent signaling pathways. These interactions have been reported in multiple forms. Posttranslational modifications, such as phosphorylation, played a major role in the regulation of gene expression and function. These covalent modifications controlled intracellular distribution, transcriptional activity, and stability of transcription factors, including Nrf2 (Kong et al. 2001; Surh 2008). Some transcription factors antagonized Nrf2 either by competing for binding to AREs or by inhibiting Nrf2 through a physical association. Small MAF proteins, BACH1, and the immediate early proteins c-FOS and FRA1 competed with Nrf2 for binding to AREs (Nguyen et al. 2000; Venugopal and Jaiswal 1996). Recently, transcriptional cross talk between Nrf2 and the aryl hydrocarbon receptor (AhR), nuclear factor-kappaB (NF-κB), p53, and Notch pathways has been reviewed in detail by several excellent publications (Nair et al. 2008; Li et al. 2008; Wakabayashi et al. 2010). Several other transcription factors, including activating transcription factor 3, proliferator-activated receptor (PPAR)g, and retinoic acid receptor a, have been reported to form inhibitory complexes with Nrf2 (Wakabayashi et al. 2010). Oxidative stress and inflammatory insult are intimately connected to each other in multistage carcinogenesis with potential cross talk between Nrf2 and NF-κB pathways (Surh 2008). Reports suggested that covalent modifications may also regulate Nrf2 after its release from Keap1. It has been observed that CREB-binding protein (CBP) induced acetylation of Nrf2 in the nucleus, resulting in binding, with basic-region leucine zipper protein(s), to the ARE and consequently in gene transcription, whereas deacetylation disengaged it from the ARE, thereby resulting in transcriptional termination and subsequently in its nuclear export (Kawai et al. 2011). NF-κB p65 subunit repressed the Nrf2-ARE pathway at transcriptional level. p65 selectively deprived CBP from Nrf2 by competitive interaction with the CH1-KIX domain of CBP, which resulted in inactivation of Nrf2.

Implication of Nrf2 in response to arsenic-induced oxidative stress

Arsenic undergoes reduction, methylation, and GSH conjugation to yield polar metabolites that are substrates for transporters. These events suggest that transcription factor(s) controlling the upregulation of antioxidant proteins, phase II xenobiotic-metabolizing enzymes, and phase III transporters should affect arsenic-mediated oxidative stress and the steady-state level of arsenic in the cells (Kumagai and Sumi 2007).

Arsenite or arsenate induced increase in Nrf2 in osteoblasts, followed by transcriptional activation of target genes encoding HO-1, Prx I, and a class of ubiquitin-binding proteins (A170) (Aono et al. 2003). As III enhanced cellular expression of Nrf2 at the transcriptional and protein levels and activated expression of Nrf2-related genes in human keratinocyte cell line, HaCaT. H2O2 was one of the mediators for the increase in Nrf2 expression and activity. Arsenic exposure caused nuclear accumulation of Nrf2 in association with downstream activation of Nrf2-mediated oxidative response genes. Simultaneously, arsenic increased the expression of a Nrf2 activity regulator, Keap1 (Pi et al. 2003). A model has been proposed to explain the binding of arsenic to different sets of Keap1 cysteine residues which regulate divergent functions in Nrf2 signal transduction. The function of arsenic–Keap1 interaction was evaluated in a reconstituted system that mimicked endogenous Nrf2 regulation. Mutation of Cys273 or Cys288 in the linker region resulted in high-level basal expression of Nrf2 protein. Mutation of Cys151 abolished Nrf2 activation by arsenic. Overexpression of Cys273A, Cys288A, or Cys151A altered the basal and arsenic-induced expression of Nrf2 target genes. The study showed an important role of Cys273 and Cys288 in the suppression of Nrf2 by Keap1 and a critical function of Cys151 in arsenic responsiveness (He and Ma 2010). Recently, Zhao et al. (2012) reported that Nrf2, Keap1, and Nrf1 interacted with each other and coordinated regulation of cellular adaptive antioxidant responses to acute inorganic arsenic exposure. This was highlighted as one of the probable causes of oxidative stress in arsenic-induced dermal toxicity and carcinogenicity. The study also provided a clear insight into the critical roles of Nrf2 and Nrf1 in defending against oxidative damage and the pathogenesis of hyperkeratosis and skin cancer. Arsenic markedly enhanced the interaction between Keap1 and Cul3, subunits of the E3 ubiquitin ligase for Nrf2, which led to impaired dynamic assembly/disassembly of the E3 ubiquitin ligase, and thereby decreased its ligase activity. Thus, arsenic inhibited Nrf2 ubiquitination and degradation and enhanced Nrf2 protein levels (Wang et al. 2008). Arsenic stabilized Nrf2 protein, extending the t1/2 of Nrf2 from 21 to 200 min by inhibiting the Keap1–Cul3-dependent ubiquitination and proteasomal turnover of Nrf2. Arsenic markedly inhibited the ubiquitination of Nrf2 without disrupting the Nrf2–Keap1–Cul3 association in the cytoplasm. In the nucleus, arsenic, but not phenolic antioxidant tBHQ, dissociated Nrf2 from Keap1 and Cul3 followed by dimerization of Nrf2 with a Maf protein (Maf G/Maf K). Nrf2 and Maf associated with the endogenous NQO1 ARE enhancer constitutively. Arsenic substantially increased the ARE occupancy by Nrf2 and Maf. In addition, Keap1 was shown to be ubiquitinated in the cytoplasm and deubiquitinated in the nucleus in the presence of arsenic without changing the protein level, implicating nuclear–cytoplasmic recycling of Keap1. This revealed that arsenic activated the Nrf2/Keap1 signaling pathway through a distinct mechanism from that of antioxidants and suggested an “onswitch” model of NQO1 transcription in which the binding of Nrf2-Maf to ARE controlled both the basal and inducible expression of NQO1 (He et al. 2006). Nrf2/small Maf heterodimers were shown to play a decisive role in the response to arsenic-mediated stress in placental cells as evidenced by binding of endogenous Nrf2/small Maf to a stress response element (StRE) recognition site and induction of HO-1 (Massrieh et al. 2006). Arsenic-induced HO-1 partly acted as a positive feedback regulator of Nrf2 activation, thereby diminishing its cytotoxicity in HepG2 cells (Abiko et al. 2010). As III promoted dissociation of Bach1 (a transcriptional repressor) from the HO-1 enhancers and increased Nrf2 binding to these elements. Bach1 cysteine residues 557 and 574 were essential for the induction of HO-1 gene in response to As III. These findings demonstrated a role for HO-1 in As III-mediated angiogenesis in vitro (Meng et al. 2010). In arsenic-exposed mouse endothelial cells (SVEC4-10), HO-1 expression has been found to be mediated through Nrf2, NF-κB, and p38 MAPK-dependent signaling pathways and serves as an upstream regulator of VEGF (Wang et al. 2012).

Nrf2-related genes, like HO-1, GCLs, and SOD, showed diminished responses in arsenic-transformed (As-TM) cells after UV exposure and exhibited reduced oxidant stress response. UV-exposed As-TM cells showed increased expression of cyclin D1 and decreased p16. UV exposure enhanced the malignant phenotype of As-TM cells. Co-carcinogenicity between UV and arsenic in skin cancer might involve adaptation to chronic arsenic exposure generally mitigating the oxidative stress response, allowing apoptotic bypass after UV and enhanced cell survival even in the face of increased UV-induced oxidative stress and increased oxidative DNA damage (Sun et al. 2011).

Study of the molecular mechanism of Nrf2 activation by arsenic demonstrates that both As(III) and MMA(III) were able to activate Nrf2 by increasing association between Keap1 and Cul3, therefore disrupting the dynamic assembly/disassembly process of the Keap1–Cul3 E3 ubiquitin ligase complex. Reduced E3 ubiquitin ligase activity led to decreased degradation of Nrf2 and activation of the Nrf2 downstream effects. Furthermore, upregulation of Nrf2 by As(III) and MMA(III) was independent of the previously identified cysteine residue C151 in Keap1, which indicated a distinct mechanism by which As(III) and MMA(III) activated Nrf2 compared to other Nrf2 inducers, such as tBHQ and SF (Wang et al. 2008). Acetylation of Nrf2 by p300/CBP in response to arsenite-induced stress augmented promoter-specific DNA binding of Nrf2 that functioned along with Keap1-mediated ubiquitination in modulation of the Nrf2-dependent antioxidant response (Sun et al. 2009).

Arsenic itself was reported to induce the Nrf2-dependent antioxidant response, although the detailed mechanism of Nrf2 induction by arsenic remains to be explored (He et al. 2006; Wang et al. 2008). Cellular Nrf2 protein levels in Chang human hepatocytes increased rapidly after 2 h of exposure, elevated significantly at 6 h, and reached the maximum at 12 h exposure of As III. The endogenous Nrf2-regulated downstream HO-1 mRNA and protein were also induced dramatically and lasted for as long as 24 h. In addition, intracellular GSH levels elevated in consistent with Nrf2 activation. Thus, it was suggested that inorganic arsenic altered cellular redox balance in hepatocytes to trigger Nrf2-regulated antioxidant responses. This reflected an adaptive cell defense mechanism against inorganic arsenic-induced liver injuries and hepatotoxicity (Li et al. 2011). Arsenic elicited both a beneficial Nrf2-dependent antioxidant response and a cell damaging effect. The toxic effects of any compound are dependent on the cellular complexity and on the multiple gene expression profile. Activation of an array of Nrf2-dependent downstream genes conditions cells for an adaptive defense response to subsequent toxic/carcinogenic insults. Other pathways that lead to cell death may also be activated. The net outcome in response to arsenic may be dictated by arsenic species, dose, and duration of arsenic exposure. Nevertheless, activation of the ARE–Nrf2–Keap1 pathway represented the initial attempt to counteract deteriorative effects induced by arsenic and to maintain cellular homeostasis. However, with high concentrations or repetitive doses of arsenic, the Nrf2-dependent defense response is outweighed by the deteriorative effects induced by arsenic, ultimately resulting in toxicity (Jiang et al. 2009).

Arsenic trioxide (ATO) was shown to have contrasting effects under in vitro and in vivo conditions. ATO had anticancer effects on both cultured oral cell carcinoma cells (OSCC) and OSCC xenografts by inhibiting cell growth, suppressing angiogenesis, and inducing apoptosis. ATO activated a silent Nrf2 pathway in cultured OSCC as shown by induction of Nrf2 downstream targets like NQO1 and HO-1. However, in OSCC xenograft tumors, Nrf2 pathway became active and ATO treatment downregulated expression of Nrf2 and Nrf2-regulated genes (Zhang et al. 2012).

Cancer prevention by targeting Nrf2 and redox signaling

An effective chemopreventive agent is able to intervene early in the process of carcinogenesis either by eliminating premalignant cells or protecting normal cells from undergoing transformation (Hail et al. 2008). Most of the chemopreventive agents can be broadly divided into two main classes, namely blocking agents and suppressing agents. Blocking agents aim to prevent carcinogens from reaching the target sites, undergoing metabolic activation, or subsequently interacting with crucial cellular macromolecules, such as DNA, RNA, and proteins (Surh 2003). Suppressing agents mostly interfere with the promotion and progression of carcinogenesis via the influence on cell proliferation, differentiation, and/or apoptosis, thereby inhibiting the premalignant and malignant transformation of initiated cells (Manson et al. 2000). One of the plausible mechanisms by which blocking agents impart their chemopreventive activity is the induction of a set of phase II detoxifying and antioxidant enzymes, including GST, NQO1, UGT, HO-1, aldehyde dehydrogenase, aldo-keto-reductase, microsomal epoxide hydrolase, γ-glutamate cysteine ligase (γ-GCL), glutathione synthetase, and γ-glutamyl transpeptidase, which act in concert to detoxify and eliminate harmful reactive intermediates formed from carcinogens (Lee and Surh 2005). As mentioned earlier, these cytoprotective enzymes are regulated through the evocation of AREs mediated by Nrf2. Therefore, the activation of Nrf2–ARE signaling pathway has been considered as an effective strategy for cancer chemoprevention (Khor et al. 2008; Surh et al. 2008; Hayes et al. 2010; Zhao et al. 2010; Baird and Dinkova-Kostova 2011). Many natural antioxidants and potential chemopreventive agents, including isothiocyanates, diallyl sulfides, indoles, terpenes, and phenolic compounds, such as tea catechins and curcuminoids, increase Nrf2 protein levels by inhibiting its turnover and induce ARE-mediated gene expression (Jeong et al. 2006). Several compounds of dietary agents in preclinical in vitro and in vivo models of various cancers have exhibited effects on the Nrf2 antioxidant system (Darvesh and Bishayee 2012). The grape polyphenol resveratrol attenuated oxidative stress and suppressed inflammatory response mediated by Nrf2 to impart chemopreventive effects against chemically induced hepatic tumorigenesis in rats (Bishayee et al. 2010). Pomegranate bioactive constituents suppressed early hepatocarcinogenic events in rats challenged with diethylnitrosamine (DENA) by a marked hepatic antioxidant activity achieved by upregulation of several antioxidant and housekeeping genes under the control of Nrf2 (Bishayee et al. 2011). Black currant anthocyanins exerted chemoprevention of DENA-initiated hepatocarcinogenesis and abrogated DENA-evoked oxidative stress by upregulation of an array of hepatic antioxidant and carcinogen-detoxifying genes through modulation of Nrf2 signaling pathway in rodents (Thoppil et al. 2012). Based on emerging studies, induction of cytoprotective enzymes by dietary chemopreventive agents through the Nrf2 pathway is currently considered to be an important and viable part of preventing cancer in the human population.

Chemoprevention of arsenic-induced carcinogenesis through modulation of Nrf2 signaling pathway

Nrf2 downstream genes like glutamyl cysteine synthetase (GCS), which modulates intracellular GSH levels, and others including GST, UGT, and MRPs contributed to the Nrf2-mediated protection against arsenic toxicity (Du et al. 2008). Sulforaphane was demonstrated to reduce intracellular arsenic concentration and increase resistance to arsenic toxicity in primary mouse hepatocytes (Shinkai et al. 2006). Human bladder epithelial cells UROtsa stably infected with Nrf2-siRNA demonstrated 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, 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 sulforaphane, whereas neither tBHQ nor sulforaphane conferred protection in the Nrf2−/−MEF cells, demonstrating that tBHQ- or sulforaphane-mediated protection against As(III)- and MMA(III)-induced toxicity depends on Nrf2 activation (Wang et al. 2007).

Pharmacological intervention using dietary factors that activated the redox-sensitive Nrf2/Keap1-ARE signaling pathway has been suggested as a promising strategy for chemoprevention of human cancer including colorectal carcinoma. Trans-cinnamic aldehyde (cinnamaldehyde, CA), the key flavor compound in cinnamon essential oil and an ethanolic extract (CE) prepared from Cinnamomum cassia bark, displayed equipotent activity as inducers of Nrf2 transcriptional activity. In human colon cancer cells (HCT116, HT29) and non-immortalized primary fetal colon cells (FHC), CA- and CE-treatment upregulated cellular protein levels of Nrf2 and established Nrf2 targets involved in the antioxidant response including HO-1 and γ-GCS, catalytic subunit. CA- and CE-pretreatment strongly upregulated cellular GSH levels and protected HCT116 cells against hydrogen peroxide-induced genotoxicity and arsenic-induced oxidative insult (Wondrak et al. 2010). Oridonin or rubesecensin A is a diterpenoid purified from the Chinese medicinal herb Rabdosia rubescens. Pretreatment of UROtsa cells with oridonin significantly enhanced the cellular redox capacity, reduced formation of ROS, and improved survival of UROtsa cells after arsenic exposure. Oridonin belonged to a novel class of Nrf2 activators. It inhibited ubiquitination and degradation of Nrf2 similar to tBHQ, resulting in stabilization of Nrf2 and activation of the Nrf2 signaling pathway (Du et al. 2008).

Phytochemicals like curcumin, catechins, and theaflavins are powerful natural antioxidants and chemopreventive agents (Bishayee and Darvesh 2010; Darvesh et al. 2012). While the clinical intersession accepts groundwater arsenic contamination as a public health problem only after the onset of the disease, interventions based on scientific knowledge for prevention of such environmental calamities may find their application to reduce the misery of the people in the endemic regions. Thus, by virtue of the antioxidant property and the repair-inducing capability of the natural compounds, an effective strategy may be formulated to combat arsenic toxicity. The work was initiated with the investigation of molecular cytogenetic effects of inorganic and organic arsenicals in normal mammalian cell lines and human lymphocytes and their amelioration by green tea and black tea polyphenols and their extracts (Table 1). The study elicited the mechanism of arsenic-induced oxidative stress and its modulation by tea bioactive constituents in Chinese hamster lung fibroblasts and in normal human lymphocytes (Sinha et al. 2003, 2005a, b, c, 2007). Various other phytochemicals, such as capsaicin, curcumin, ellagic acid, fisetin, quercetin, resveratrol, and rutin, have been found to exert protection of arsenic-induced toxicity in Chinese hamster lung fibroblasts by antioxidant mechanisms (Roy et al. 2008). Detailed molecular mechanisms involved in mitigation of arsenic-induced imbalance of redox homeostasis by antioxidant activity of tea polyphenols were observed in Swiss albino mice (Sinha et al. 2010). Green and black tea polyphenols also exhibited efficacy in imparting protection against arsenic-induced oxidative DNA adducts and inhibition of DNA repair in Swiss albino mice (Sinha and Roy 2011). Apart from tea polyphenols, curcumin also proved to be effective against toxic impact of arsenic generated in Swiss albino mice (Biswas et al. 2010b). An interesting field study was made to ascertain the possibility of using curcumin as a chemopreventive intervention against the adverse effects of chronic arsenic exposure to humans through groundwater contamination (Sinha et al. 2009; Biswas et al. 2010a). A pilot study was made to exhibit the protective effect of curcumin against arsenic-induced inhibition of DNA repair pathways in a field trial (Roy et al. 2011).

Table 1 Amelioration of arsenic-induced cytogenetic effects, oxidative stress, and repair inhibition by various phytoconstituents
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As summarized in Table 1, tea polyphenols as well as curcumin have been found to mitigate arsenic-induced cytogenetic effects, oxidative stress, and repair inhibition in different model systems. Moreover, tea catechins, curcumin, resveratrol, as well as organosulfur compounds possess Nrf2 modulatory properties, which have been recently reviewed in detail by Darvesh and Bishayee (2012). These compounds may have future implications in mitigation of arsenic toxicity with the improvisation of Nrf2 redox regulation.

Future indications in combating arsenic-induced carcinogenicity by improvising Nrf2 signaling

The present-day research has highlighted Nrf2-mediated wide-spectrum regulation of cytoprotective and transcriptional response leading to prevention of damage to DNA, proteins, and lipids; recognition, repair, and removal of macromolecular damage; and tissue renewal following toxic assaults. Literature review exhibited enhanced frequency of tumor burden in Nrf2-disrupted mice compared to wild-type models of inflammation and colon cancer, bladder cancer, lung disease and cancer, stomach cancer, mammary cancer, skin cancer, and hepatocarcinogenesis (Slocum and Kensler 2011). Activation of Nrf2 represented a beneficial effect of cells to confer protection against arsenic toxicity, but paradoxically arsenic was reported to activate the Nrf2 pathway. It was speculated that the stress-inducible transcription factor Nrf2 contributed significantly to the hormesis or adaptive response. Activation of the ARE–Nrf2–Keap1 pathway by As(III) at either low or high concentration is beneficial and is likely to be an attempt of cells to counteract the damaging effects, although the protective mechanism of the Nrf2 pathway may be masked by cell death effects at high concentrations of arsenic. Response to a toxicant in accordance with the complexity of gene expression profile is dependent on batteries of genes working in concert. The activation of an array of Nrf2 downstream target genes preconditions cells to a defensive mode for toxic/carcinogenic insults. Several pathways that lead to different responses are activated by arsenic exposure. The net outcome is dictated by species, dose of arsenic, and duration of exposure. With high concentrations or repetitive doses of arsenic exposure, the Nrf2-dependent defense response is outweighed by the deteriorative effects induced by arsenic, resulting in acute toxicity or cell transformation. Therefore, it is believed that specific and potent activation of Nrf2 to elicit cellular hormesis by an agent with low toxicity, such as sulforaphane, should be a great strategy to combat arsenic-induced harmful effects (Wang et al. 2007). A simplified schematic representation has been shown to elucidate the basal status and induced profile of Nrf2 under the influence of arsenic and phytochemicals (Fig. 1).

Fig. 1
figure 1

The differential status of Nrf2 under basal state and induced state has been represented. a Cytosolic sequestration of Nrf2 by Keap 1 and proteasomal degradation under basal condition; b induction of Nrf2 under the hormetic influence of arsenic, blocking of proteasomal degradation of Nrf2, and nuclear translocation followed by genetic transcription of Nrf2 downstream targets containing ARE promoters; and c induction of Nrf2 by phytochemicals involving several signal transduction pathways with subsequent phosphorylation and dissociation of Keap1 from Cul3, nuclear translocation of Nrf2 followed by genetic transcription of Nrf2 downstream targets containing ARE promoters

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Recent evidences suggested that various dietary phytoconstituents afforded chemoprevention of hepatocarcinogenesis in preclinical animal models possibly through potent antioxidant activity achieved by upregulation of several housekeeping genes under the control of Nrf2 without toxicity (Bishayee et al. 2010, 2011; Thoppil et al. 2012). Therefore, identification, validation, and optimization of new Nrf2 activators are essential for the development of effective dietary supplements or therapeutic drugs that can be used to boost the Nrf2-dependent adaptive system to protect humans from various environmental insults. Targeting the activation of Nrf2 may prove to be a superior approach for designing and developing more effective chemopreventive drugs. Diet-based Nrf2 inducers offer greater advantages over therapeutic drugs due to their general acceptance, low toxicity, and cost-effectiveness. Thus, inclusion of phytochemicals to boost the Nrf2-dependent antioxidant response in high-risk arsenic-exposed populations is an excellent choice to combat arsenic-induced toxicity and thereby carcinogenicity. Combating arsenic-mediated cancers through the selective induction of Nrf2 by dietary phytoconstituents may be considered a viable approach for cancer control in several underdeveloped and developing countries where a large population is at a significant risk of developing arsenic-induced skin cancer.