Int. J. Environ. Res. Public Health 2012, 9 485 Cellular thiols such as glutathione (GSH) and metallothionein are important in maintaining the integrity of cytoskeletal structures and functions [21,22]. Interaction with cellular thiols is the most important biological property of As3+ [12,27]. Thus, we further examined the levels of other cellular thiols such as metallothionein (I and II isoforms) and thiol-related protein such as the heavy chain of -GCS, a rate limiting enzyme for GSH synthesis [28], in As3+-exposed RFL6 cells. As shown (Figure 8), treatment of cells with As3+ at indicated concentrations induced a dose-dependent elevation of cellular levels of metallothionein [Figure 8(A)] and the -GCS heavy chain [Figure 8(B)]. Thus, RFL6 cells exhibited a sensitive response to As3+ exposure in terms of altered cellular thiol homeostasis. Figure 8. As3+ enhanced expression of metallothionein and -GCS. Growth-arrested RFL6 cells were treated with As3+ at indicated concentrations for 24 h and processed for Western blot analysis. (A) metallothionein (I and II); (B) -GCS heavy chain; actin as an internal control. Taxol is an MT stabilizing drug [13]. To assess taxol protective effect on As3+-induced MT injury, we examined MT organization in As3+-treated RFL6 cells in the presence or absence of taxol. In comparison to the control [Figure 9(A)], treatment of cells with taxol at 20 µM induced MT bundling [Figure 9(B)]. As expected, 20 µM As3+ exposure for 24 h induced severe loss of cellular MTs [Figure 9(C)]. In contrast, co-incubation with 20 µM taxol prevented As3+-induced MT disruption [Figure 9(D)]. In addition, taxol also blocked disassembly of MTs induced by NEM (25 µM), a sulfhydryl group binding agent [21], which was used as a positive control in this experiment [comparing Figure 9(F), NEM + Taxol with Figure 9(E), NEM alone]. Thus, taxol antagonizes disassembly of MTs by thiol-bound agents such as As3+ and NEM. As3+ damage to the MT organization may be facilitated by alterations in MAPs which are important in maintaining the structural and functional integrity of MTs [29]. Because MTs are very sensitive to Ca2+, proteins released by Ca2+ extraction of the detergent resistant MTs of cells, by definition, contain tubulins and MAPs [25]. As analyzed by two-dimension (2-D) gels, quiescent RFL6 cells treated with 5 µg/mL nocodazole, a MT disassembly agent, a positive control in this study, for 3 h, displayed marked reduction of MT proteins in association with disappearing several MAPs such as 50/6.5, 53/6.5, 60/6.4, 62/6.3 and 86/6.5 (apparent MW in kDa/isoelectric point) indicated by arrowheads