Int. J. Environ. Res. Public Health 2012, 9 481 Figure 2. Deficient spindle MTs and cell division aberrations in As3+-treated cells. RFL6 cells on coverslips were incubated in 10% FBS medium without (a–e) or with (f–j) 10 µM As3+ for 24 h. Cell division stages and aberrations were examined by visualization of the in situ chromosomes stained with propidium iodine (red) and spindle MT stained with FITC-conjugated anti-tubulin antibody (green). Note: Cytoplasmic FITC-labeled MTs are green; however, since spindle MTs overlap with DNA staining in red, they appear as yellow structures. a, prophase; b, prometaphase; c, metaphase; d, anaphase; e, telophase; f, chromosome lagging with a defective spindle at metaphase; g, chromosome scattering in the absence of spindle; h, chromosome clustering and scattering in the absence of spindle; i, chromosome lagging with a defective spindle at anaphase; j, apoptosis of a cell with aberrant division. Figure 3. As3+ effects on expressions of tubulin proteins and mRNAs in the cell model. (A) As3+ enhanced the free tubulin pool as a result of depolymerization of MTs. Growth-arrested RFL6 cells were treated with As3+ at indicated concentrations for 24 h. Cytoskeletal and cytosolic fractions were isolated, and analyzed by Western blot. β-tubulin, 50 kDa; actin, 43 kDa used as a reference. (B) As3+ inhibition of steady-state βI tubulin mRNA levels. Cells were treated with As3+ as indicated above. Total RNA extracted was assessed for steady-state βI tubulin mRNA levels by the RT-PCR. GAPDH mRNA levels were used as an internal control. M indicates a DNA molecular ladder. To elucidate As3+ damage to the lung MTs, a small animal model study was performed to visualize tubulin protein distribution by immunohistochemistry and to assay for steady-state I tubulin mRNA expression by the RT-PCR in control and As3+-dosed lungs. As shown [Figure 4(A)],