Int. J. Environ. Res. Public Health 2012, 9 482 immunoperoxidase staining of the lung tissue indicated markedly weakened tubulin signals, i.e., peroxidase-labeled brown-black color materials, in As3+-treated lung tissues [Figure 4(A2)] in comparison to the control [Figure 4(A1)]. Figure 4(A3) is a negative control tissue slice from control rats, which was incubated in the absence of the primary antibody against -tubulin. Notably, As3+ exposure apparently induced discontinuity of tubulin-rich epithelial cells in small bronchioles (arrow). Furthermore, RT-PCR assays indicated that As3+-dosed lungs displayed decreased βI tubulin mRNA levels amounting to 53%, 48% and 20% of the control in As3+-exposed Rat#1, Rat#2 and Rat#3, respectively [Figure 4(B)]. Thus, As3+ inhibited pulmonary tubulin expression at protein and mRNA levels in vivo. Figure 4. Inhibition of β-tubulin expression at protein and mRNA levels in As3+-exposed lungs. Rats (150 g body weight) were intratracheally instilled with 520–530 µg NaAsO2 in 100 µL physiological saline according to 2.02 mg As/kg body weight once a week for 5 weeks. Control rats received saline only. Rats were killed a week after the last instillation. Lung tissues were processed for immunohistochemical staining and total RNA extraction. (A) β-Tubulin protein distribution. The tubulin protein in control and As3+-dosed lung sections was stained with the antibody against -tubulin and the streptavidin-HRP system. (A1) control; (A2) As3+ exposed lung; (A3) a negative control without the primary antibody treatment. a, alveolus; b, bronchioles; arrow indicates discontinuity of tubulin-rich epithelial cells in small bronchioles. (B) I-tubulin mRNA levels revealed by the RT-PCR. RC, control rat; RAs1, As3+ treated Rat#1; RAs2, As3+-treated Rat#2; RAs3, As3+-treated Rat#3 (note: single doses for Rat #1, #2 and #3 were 520, 525 and 530 µg NaAsO2 , respectively, dependent on the body weight). M, a DNA molecular ladder. To identify characteristics of the interaction of As3+ with tubulin proteins, we assessed its effects on the MT polymerization in vitro. Bovine brain MT proteins containing tubulins and MAPs were purified through two cycles of temperature dependent assembly and disassembly [17]. MT assembly in vitro was monitored by turbidity assays at A350 nm (Figure 5) and confirmed by electron microscopy (EM) (Figure 6) as described [16,17]. In a typical turbidity assay at 25 C [the black curve, in Figure 5(A)], assembly of MT proteins (0.8 mg/mL) progressed steadily with an initial leg (two min) followed by an elongation phase (2–30 min) and a final plateau indicating the reaction equilibrium (30–32 min). EM examination of samples taken at 30 min revealed the typical morphology of in vitro assembled MTs [Figure 6(B)]. No assembly of MTs was evident at time 0 [Figure 6(A)]. Incubation with As3+ induced a dose-dependent inhibition of MT assembly as evidenced by enhancement of the