Int. J. Environ. Res. Public Health 2012, 9 483 initial leg, decreases in the elongation rates and the plateau levels. For example, the average elongation rates at 12 min of the reaction (the turbidity values/time) were decreased to 50.0%, and 10.4% of the control at 20 (the green curve) and 40 (the blue curve) µM of As3+, respectively [Figure 5(A)]. The inhibition of MT assembly by As3+ was further confirmed by EM which showed significant reduction in the number of MT fragments in the presence of 40 µM As3+ [Figure 6(C)], amounting to 50% of the control [Figure 6(B)]. Importantly, addition of 1 mM dithiothreitol (DTT), a thiol reducing agent, to the reaction mixture strongly abolished the As3+ inhibitory effect on MT assembly as shown in the turbidity [the pink curve in Figure 5(B)] and EM assays [Figure 6(D)] reaching 95% (the turbidity assay) and 108% (the EM assay) of corresponding controls, respectively. Thus, As3+ inhibited MT polymerization in vitro and results provide a critical clue for the direct interaction of As3+ with tubulin -SH groups. Figure 5. As3+-inhibition of MT protein polymerization in vitro as revealed by the turbidity assay. The in vitro polymerization of MT proteins (0.8 mg/mL) in 0.1 M Pipes buffer, pH 6.6, containing 0.15 mM Mg2+, 0.15 mM EGTA and 500 µM GTP was monitored by turbidity readings at A350nm at 25 °C in the absence or presence of As3+ at indicated concentrations. Curves represent the time course of tubulin assembly under different conditions. (A) dose-dependent inhibition by As3+ of MT assembly. the black curve, control; the green curve, 20 µM As3+; the blue curve, 40 µM As3+. (B) DTT effects on As3+ inhibition of MT assembly. DTT (1 mM) was added after preincubation of polymerization mixture in the presence of As3+ for three min. the black curve, control; the brown curve, 1 mM DTT; the blue curve, 40 µM As3+; the pink curve, 40 µM As3+ plus 1 mM DTT. To further assess As3+ binding to tubulin protein -SH groups, we also examined the competition of As3+ with N-ethylmaleimide (NEM), a sulfhyryl group reagent [21], for tubulin binding. “Pure” tubulin free of MAP (1.5 mg/mL) prepared from bovine brain was pretreated with As3+ at indicated doses for 2 h at 0 C, mixed with [3H]NEM (2 µCi/mL), and incubated for an additional 2 h at 37 C. Proteins were precipitated with 5% TCA and filtered. Radioactivities associated with collected proteins on the membrane were measured by -counting. As shown (Figure 7), As3+ decreased the binding of