Int. J. Environ. Res. Public Health 2012, 9 480 Figure 1. As3+ induced disassembly of MTs in RFL6 cells. Growth-arrested RFL6 cells on coverslips were treated without or with As3+ at indicated concentrations for 24 h, and then immunofluorescently stained for visualization of MTs. (A) control; (B) 5 µM As3+; (C) 20 µM As3+. Since spindle MTs are intrinsically involved in chromosomal organization during cell mitosis [13,14], As3+ may perturb spindle MTs inducing abnormal cell division. To test this, we have examined cell division aberrations by fluorescently double-staining spindle MTs and chromosomes in situ in As3+-treated RFL6 cells. As shown (Figure 2), exposure of RFL6 cells to 10 µM As3+ for 24 h resulted in the defect in or the absence of spindle MTs in 34% of proliferating cells (200 cells counted) accompanied by chromosomal lagging, scattering, clustering, etc., in 22% of proliferating cells (200 cells counted) [Figure 2(f–j)], in sharp contrast to those in control cells (only 2% of proliferating cells counted with spindle deficiency and 1.5% of proliferating cells counted with chromosomal disorientations). Figure 2(a–e) show normal chromosome patterns in different phases of the cell division. These results indicate that chromosomal disorientations are closely linked to As3+-induced damage to spindle MTs. As3+-induced MT disassembly (Figure 1) should decrease the polymer MT and increase the free tubulin pool. To test this, As3+-treated cells were treated with 0.3% NP40 in the PM2G buffer to isolate cytosolic and detergent-resistant cytoskeleton fractions [21]. Western blot analysis indicated that in comparison to the actin control, RFL6 cells exposed to As3+ exhibited a dose-dependent decrease in polymer MT proteins and increase in the free tubulin pool [Figure 3(A)]. The density assays indicated that the 50% decrease in MT polymer proteins and increase in the free tubulin pool occurred in cells exposed to 5 µM As3+. These results are consistent with morphological loss of MTs and increase of free tubulin mass in these cells as revealed by fluorescent microscopy. Five β-tubulin genes encode highly homologous proteins, class I–V [26]. The rat class I β-tubulin (βI-tubulin) gene was selected as a sample to elucidate As3+ modulation of tubulin transcription. As shown by the RT-PCR assay [Figure 3(B)], the steady-state I tubulin mRNA levels in cells treated with 5, 10 and 20 µM As3+ were reduced to 66%, 26% and 6% of the control, respectively. Thus, As3+ depolymerization of MTs was associated with inhibition of tubulin mRNAs.