Int. J. Environ. Res. Public Health 2012, 9 477 killed 1 week after the last instillation. For immunohistochemistry, lungs removed from four rats of each group were fixed with 0.2% glutaraldehyde and 4% paraformaldehyde in 0.1 M phosphate buffer, pH 7.4. Lung tissues were embedded in paraffin. Sections of 5 m in thickness were immunohistochemically stained to visualize tubulin distribution in lungs with the anti-tubulin antibody and the streptavidin-HRP system according to the procedure provided by the manufacturer (KPL, Inc., Gaithersburg, MD, USA). For total RNA extraction, lungs in other four rats of each group were perfused with physiological saline via the pulmonary artery. The minced lung tissues were homogenized in TRIzol reagent (Invitrogen) and total RNA were extracted with phenol-chloroform as described [23]. 2.5. Purification of MT Proteins MT proteins containing tubulins and MAPs were purified from calf brain through two cycles of temperature-dependent assembly-disassembly as described in our previous publications [16,17]. The MT protein pellet was dissolved in a PME buffer (0.1 M Pipes, pH 6.6, 1 mM MgCl2 and 1 mM EGTA) and aliquots of this MT protein stock were stored at −80 °C until their use in experiments. Pure tubulin free of MAP was prepared by passing the twice-cycled MT proteins through a Whatman P11 phosphocellulose column as described [24]. 2.6. Turbidity Assay The original MT protein stock was diluted with the 0.1 M Pipes buffer, pH 6.6, to yield a final concentration of 0.8 mg/mL with 0.15 mM Mg2+ and 0.15 mM EGTA. MT polymerization in vitro was started by the addition of 500 µM GTP and monitored by turbidimetry at A350 nm at 25 C using a Perkin-Elmer Lambda 3B spectrophotometer equipped with a chart recorder [16,17]. To assess effects of As3+ on MT assembly in vitro, various concentrations of As3+ were added to the reaction mixtures for 5 min prior to addition of GTP. To observe DTT influence on As3+-induced effects on MT polymerization, As3+, DTT (1 mM) and final GTP were added to the reaction mixture in this order with the 3-min interval between additions to allow the interaction of As3+ with MT proteins. 2.7. Electron Microscopy (EM) EM examination of assembled MT polymers was carried out in conjunction with turbidity assays as described [16]. When the MT assembly reaction was approaching its plateau, a 10 µL-drop of the reaction mixture was removed from the cuvette and placed on glow-discharged carbon formvar-coated copper grids (300 mesh) for five min and successively replaced by cytochrome c (1 mg/mL, from Sigma) and distilled water. The samples were stained with filtered 1% uranyl acetate for 3 min, blotted, air dried, and examined with a Philips CM12 transmission electron microscope. All EM images were recorded on SO-163 film. MT numbers on three photo prints with the same size and magnification were counted for each sample and results are expressed as % of the control. 2.8. Tubulin Sulfhydryl (-SH) Assay Tubulin -SH groups were determined as described in our previous publication [22]. This assay is based on covalent incorporation of [3H]NEM, a specific -SH group binding agent, to protein -SH