Int. J. Environ. Res. Public Health 2012, 9 489 mechanism for As3+ toxicity [12,27]. Our previous and present studies showing As3+ depolymerization of MTs indicated that tubilin, a critical cell structure protein, is a target for As3+ insult. A dimeric tubulin molecule contains 20 cysteine residues (12 for α and eight for β), most of which are biochemically accessible. Maintenance of certain tubulin -SH groups in the reduced form is crucial for MT polymerization since their loss induces MT disassembly [32,33]. One such “assembly-critical” -SH group, for example Cys239, has been identified in -tubulin [33,34]. Cys239 mediates the lateral interaction of tubulin molecules required for MT assembly [33]. Thus, tubulin -SH groups are of a critical stabilizer for MTs. In this report, to identify the mechanism for As3+ depolymerization of MTs, we performed the in vitro MT polymerization assay using purified tubulins isolated from bovine brain. As3+ incubation with tubulin proteins inhibited MT assembly in vitro and DTT, a thiol-reducing agent, reversed the As3+ suppression of MT assembly (Figures 5 and 6) supporting As3+ interaction with tubulin -SH groups. One should be noted. As3+ concentrations used in this in vitro MT assembly study were higher than those used in cell culture studies. This is most likely due to: (1) low concentrations of MT proteins in cultured cells, and (2) more abundant MAPs in the bovine brain tissue compared to non-neural cells, e.g., fibroblasts [35]. As3+ doses used in present studies, i.e., 5–20 µM (0.374–1.498 ppm) for RFL6 cells, 10–40 µM (0.748–2.996 ppm) for in vitro tubulin polymerization assays, and 2.02 ppm for the rat animal model, are relevant to human exposure situation. The As content in the well drinking water of some areas in India reaches 3.4 ppm [36]. Furthermore, using the in vitro [3H]NEM binding competition assay (Figure 7) we demonstrated again As3+ binding to tubulin -SH groups. Decreases in [3H]NEM association with tubulins in the protein mixture preincubated with As3+ provided more direct evidence for As3+ targeting -SH groups of MT proteins initiating the MT injury cascade. More importantly, As exposure stimulates the formation of the reactive oxygen species (ROS) and activation of oxidative stress signaling such as the NF-kB pathway in the biological system [37] which act as an amplification system to further facilitate oxidation of sulfhydryl groups on tubulins and cell injuries. As reported, cells treated with hydrogen peroxide exhibited disruption of the MT cytoskeleton [38]. Our previous studies have shown As3+-induced a biphasic response in cellular glutathione (GSH), i.e., an initial decrease followed by a full restoration and overshooting. Inhibition of cellular GSH by buthionine sulfoximine (BSO), an inhibitor of GSH biosynthesis, apparently increased cell sensitivity to As3+ toxicity [21]. Since As3+ targets cellular thiols we also examined other thiol-containing molecules such as metallothionein as well as -GCS, a rate limiting GSH synthetase [28], in cell response to As3+. In this report, we illustrated As3+ enhanced expression of metallothionein and -GCS at the protein levels accompanied by depolymerization of MT. The metallothionein molecule with 20 Cys residues can bind six As3+ while GSH binds As3+ to form As(SG)3 complex which favors transferring of As3+ to other thiol containing components [39,40]. Increased expression of -GCS as an upstream event is required for downstream enhancement of cellular GSH in cells treated with As3+ [21]. The metallothionein gene contains multiple copies of the metal responsive element (MRE) in its promoter region. MRE-binding transcription factor-1 (MTF-1), a zinc (Zn) finger protein, interacts with the MRE transactivating the metallothionein gene [41]. Zn also binds to tubulin proteins stimulating MT assembly in vitro. Tubulins isolated from brains of Zn-deficient animals showed an impaired ability for polymerization of MTs [42]. As3+ upregulated methallothioneins possibly by its binding to sulfhydryls of tubulins or other proteins releasing bound Zn ions that in turn enhance MTF-1 affinity for MRE containing genes such as metallothionein [41]. The NF-E2-related factor-2 (Nrf2)