Arsenic (As) is an abundant metalloid element in the Earth’s crust. Its compounds are widely used in industries, agricultures, and medicines [1]. In addition to occupational contact, consumption of contaminated drinking water is a major source of As exposure for humans. Most As in drinking water exists as inorganic forms released from either certain types of rock or man’s past activities [1,2]. An estimated 100–140 million people around the world are suffering from chronic As poisoning [3,4]. Inhaled or ingested As can be rapidly absorbed, enter the bloodstream, and distribute throughout the body [5,6]. The lung is one of the major target organs of As, either from contaminated air or from polluted water, and leading to the development of bronchiectasis, bronchitis, chronic obstructive pulmonary disease (COPD), and malignancies [2,7]. Environmental exposure to As is generally in the form of either arsenite (As³⁺) or arsenate (As⁵⁺) [6,8,9], with As³⁺ being more potent in its biological effects than As⁵⁺ [9,10]. As³⁺ is the predominant form in drinking water from deep (anaerobic) wells while As⁵⁺ predominates under aerobic conditions. Any As⁵⁺ in the water is rapidly reduced to As³⁺ as it enters cells [8,9]. Since As³⁺ displays high affinity for -SH groups perturbing essential proteins [11,12], As³⁺ compounds are of major human health concern. However, the precise mechanisms leading to lung damages and cancinogenesis by As³⁺ remain unclear and should be further investigated.

Microtubules (MTs) are a highly organized network of filamentous structures in the cytoplasm of eukaryotic cells [13]. The principal protein of MTs is a heterodimer consisting of tightly linked globular peptides, i.e., α- and β-tubulins, which polymerize into long, hollow, cylindrical filaments within the cell. In addition, a third subunit of MTs is γ-tubulin, functioning in the nucleation of mitotic spindle MTs. The most prominent feature of MTs is a rapid exchange between tubulin subunits and polymers. Lung cells contain abundant MTs which play a critical role in maintaining cellular architecture and internal organization, and in regulating cell shape, motility, transport, division, etc. [14]. Many drugs and toxicants can interact with cytoskeletal MTs [13,15]. For example, colchicine and vinblastine disassemble MTs and block mitotic spindle formation. In contrast, taxol promotes tubulin assembly and stabilizes MTs. Notably, metal ions display important modulating effects on MT dynamic. As shown by us and others, Mg²⁺, Be²⁺ and Ni²⁺ promote MT assembly while Ca²⁺, Cu²⁺, Cd²⁺, Hg²⁺, Cr⁶⁺ and As³⁺ cause MT disassembly [16,17,18,19,20,21]. Thus, MT injuries may be a critical manifestation of the pathogenesis of a number of toxic agents.

Our previous studies have shown As³⁺ disassembly of MTs in cultured Swiss 3T3 cells [21]. To further understand mechanisms for cellular toxicity of As in the lung, we examined effects of As³⁺, a major toxic form of As in the nature, on MT assembly in vitro, in cultured rat lung fibroblasts (RFL6) and in lung tissues of the rat animal model. Results demonstrated that As³⁺ targets tubulin sulfhydryls and MT-associated proteins (MAPs) disrupting the MT organization in tubulin polymerization in vitro and in rat lung cells in vitro and in vivo.

To elucidate As³⁺ effects on cellular MT organization, growth arrested RFL6 cells on coverslips were incubated with or without freshly prepared As³⁺ solution at final concentrations as indicated. Cellular MT distribution was revealed by immunocytochemistry [21]. As shown in Figure 1(A), growth-arrested RFL6 cells contain an intricate, highly organized network of MTs that radiate outward from the MT organizing center (MTOC) in the perinuclear region to the cell periphery. Exposure of cells to 5 µM As³⁺ for 24 h induced partial loss of MTs and no-organized tubulins with brightly staining accumulated in the perinuclear area where the MTOC is generally located [Figure 1(B)]. Treatment of cells with 20 µM As³⁺ for 24 h resulted in severe loss of MTs. Instead, mast cells exhibited high-density of depolymerized tubulin mass concentrated in the central area of cells [Figure 1(C)]. Notably, the maximum doses of As³⁺ (20 µM) that disrupted the MT organization in the cell model did not affect cell viability seriously (about 10% decrease) as assayed by trypan blue staining (data not shown). Morphological alterations in MTs were reversible after removal of As³⁺. These studies indicated that the MT cytoskeleton is an important cellular target for As³⁺ insult.

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 βI 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.

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 βI tubulin mRNA expression by the RT-PCR in control and As³⁺-dosed lungs. As shown [Figure 4(A)], 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 β-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.

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 A_{350 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 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.

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 β-counting. As shown (Figure 7), As³⁺ decreased the binding of [³H]NEM to tubulin with 50% inhibition occurring at 23 µM. This straightforward experiment shows As³⁺ binding to tubulin through -SH groups, initiating the cascade of MT disruption events.

Cellular thiols such as glutathione (GSH) and metallothionein are important in maintaining the integrity of cytoskeletal structures and functions [21,22]. Interaction with cellular thiols is the most important biological property of As³⁺ [12,27]. Thus, we further examined the levels of other cellular thiols such as metallothionein (I and II isoforms) and thiol-related protein such as the heavy chain of γ-GCS, a rate limiting enzyme for GSH synthesis [28], in As³⁺-exposed RFL6 cells. As shown (Figure 8), treatment of cells with As³⁺ at indicated concentrations induced a dose-dependent elevation of cellular levels of metallothionein [Figure 8(A)] and the γ-GCS heavy chain [Figure 8(B)]. Thus, RFL6 cells exhibited a sensitive response to As³⁺ exposure in terms of altered cellular thiol homeostasis.

Taxol is an MT stabilizing drug [13]. To assess taxol protective effect on As³⁺-induced MT injury, we examined MT organization in As³⁺-treated RFL6 cells in the presence or absence of taxol. In comparison to the control [Figure 9(A)], treatment of cells with taxol at 20 µM induced MT bundling [Figure 9(B)]. As expected, 20 µM As³⁺ exposure for 24 h induced severe loss of cellular MTs [Figure 9(C)]. In contrast, co-incubation with 20 µM taxol prevented As³⁺-induced MT disruption [Figure 9(D)]. In addition, taxol also blocked disassembly of MTs induced by NEM (25 µM), a sulfhydryl group binding agent [21], which was used as a positive control in this experiment [comparing Figure 9(F), NEM + Taxol with Figure 9(E), NEM alone]. Thus, taxol antagonizes disassembly of MTs by thiol-bound agents such as As³⁺ and NEM.

As³⁺ damage to the MT organization may be facilitated by alterations in MAPs which are important in maintaining the structural and functional integrity of MTs [29]. Because MTs are very sensitive to Ca²⁺, proteins released by Ca²⁺ extraction of the detergent resistant MTs of cells, by definition, contain tubulins and MAPs [25]. As analyzed by two-dimension (2-D) gels, quiescent RFL6 cells treated with 5 µg/mL nocodazole, a MT disassembly agent, a positive control in this study, for 3 h, displayed marked reduction of MT proteins in association with disappearing several MAPs such as 50/6.5, 53/6.5, 60/6.4, 62/6.3 and 86/6.5 (apparent MW in kDa/isoelectric point) indicated by arrowheads [compare Figure 10(D) with Figure 10(C)]. Similarly, cells incubated with 10 µM As³⁺ for 24 h exhibited a decrease in α and β tubulins and those MAPs sensitive to nocodazole [arrowheads in Figure 10(B)] in comparison to the control in the absence of As³⁺ treatment [Figure 10(A)]. Note that the internal control of actin was not significantly changed. Quantitative densitometric analysis indicated that the integrated intensities of MAPs 50/6.5, 53/6.5, 60/6.4, 62/6.3 and 86/6.5 were decreased to 44%, 49%, 15%, 48%, and 12% of the control, respectively. In view of the critical role of MAPs in the MT stability [29], decreases in the levels of several MAPs would contribute to MT instability favoring their disassembly.

Our previous studies have shown As³⁺ perturbation of the cytoskeletal organization and cellular glutathione homeostasis in Swiss 3T 3 cells [21]. The lung is a major target organ for arsenic. In this report, we further evaluated As³⁺ effects on the organization of MTs, a critical element of the cytoskeleton, in vitro, in cultured rat fetal lung fibroblasts and in lungs of the animal model. RFL6 cells were selected as an in vitro model in this study because they display a typical MT morphology sensitive to As³⁺ injury (Figure 1). Results indicated that As³⁺ disrupted MT assembly by targeting tubulin sulfhytryl groups and MAPs, two major stabilizers of MTs. Our findings showing As³⁺-elicited biochemical pathology of MTs are summarized in Table 1.

Arsenic is a metalloid element existing in inorganic and organic forms and in different oxidative states (−3, 0, +3 and +5) which distribute in water, soil and air from nature and anthropogenic sources. High-level exposure to arsenic is involved in workers in metal smelting, wood preservation and semiconductor industries [1]. The acute inhalation of arsenic dust or fume resulted in gastrointestinal effects, and central and peripheral nervous disorders such as vomiting, diarrhea, bloody urine, anuria, shock, convulsion coma and death [1,30]. Chronic exposure to arsenic in humans, by inhalation and oral routes via contaminated water, food and air, are strongly associated with anemia, peripheral neuropathy, lesions and carcinogenesis in the lung, liver, bladder, skin, kidney, etc. [1]. As³⁺ is 60-times more toxic than As⁵⁺ [31]. As³⁺ has a high affinity for sulfhydryl groups in proteins, such as enzymes, receptors or coenzymes. Binding of As³⁺ to critical thiol groups of proteins may be important mechanism for As³⁺ toxicity [12,27]. Our previous and present studies showing As³⁺ depolymerization of MTs indicated that tubilin, a critical cell structure protein, is a target for As³⁺ insult.

A dimeric tubulin molecule contains 20 cysteine residues (12 for α and eight for β), most of which are biochemically accessible. Maintenance of certain tubulin -SH groups in the reduced form is crucial for MT polymerization since their loss induces MT disassembly [32,33]. One such “assembly-critical” -SH group, for example Cys₂₃₉, has been identified in β-tubulin [33,34]. Cys₂₃₉ mediates the lateral interaction of tubulin molecules required for MT assembly [33]. Thus, tubulin -SH groups are of a critical stabilizer for MTs. In this report, to identify the mechanism for As³⁺ depolymerization of MTs, we performed the in vitro MT polymerization assay using purified tubulins isolated from bovine brain. As³⁺ incubation with tubulin proteins inhibited MT assembly in vitro and DTT, a thiol-reducing agent, reversed the As³⁺ suppression of MT assembly (Figure 5 and Figure 6) supporting As³⁺ interaction with tubulin -SH groups. One should be noted. As³⁺ concentrations used in this in vitro MT assembly study were higher than those used in cell culture studies. This is most likely due to: (1) low concentrations of MT proteins in cultured cells, and (2) more abundant MAPs in the bovine brain tissue compared to non-neural cells, e.g., fibroblasts [35]. As³⁺ doses used in present studies, i.e., 5–20 µM (0.374–1.498 ppm) for RFL6 cells, 10–40 µM (0.748–2.996 ppm) for in vitro tubulin polymerization assays, and 2.02 ppm for the rat animal model, are relevant to human exposure situation. The As content in the well drinking water of some areas in India reaches 3.4 ppm [36]. Furthermore, using the in vitro [³H]NEM binding competition assay (Figure 7) we demonstrated again As³⁺ binding to tubulin -SH groups. Decreases in [³H]NEM association with tubulins in the protein mixture preincubated with As³⁺ provided more direct evidence for As³⁺ targeting -SH groups of MT proteins initiating the MT injury cascade. More importantly, As exposure stimulates the formation of the reactive oxygen species (ROS) and activation of oxidative stress signaling such as the NF-kB pathway in the biological system [37] which act as an amplification system to further facilitate oxidation of sulfhydryl groups on tubulins and cell injuries. As reported, cells treated with hydrogen peroxide exhibited disruption of the MT cytoskeleton [38].

Our previous studies have shown As³⁺-induced a biphasic response in cellular glutathione (GSH), i.e., an initial decrease followed by a full restoration and overshooting. Inhibition of cellular GSH by buthionine sulfoximine (BSO), an inhibitor of GSH biosynthesis, apparently increased cell sensitivity to As³⁺ toxicity [21]. Since As³⁺ targets cellular thiols we also examined other thiol-containing molecules such as metallothionein as well as γ-GCS, a rate limiting GSH synthetase [28], in cell response to As³⁺. In this report, we illustrated As³⁺ enhanced expression of metallothionein and γ-GCS at the protein levels accompanied by depolymerization of MT. The metallothionein molecule with 20 Cys residues can bind six As³⁺ while GSH binds As³⁺ to form As(SG)3 complex which favors transferring of As³⁺ to other thiol containing components [39,40]. Increased expression of γ-GCS as an upstream event is required for downstream enhancement of cellular GSH in cells treated with As³⁺ [21]. The metallothionein gene contains multiple copies of the metal responsive element (MRE) in its promoter region. MRE-binding transcription factor-1 (MTF-1), a zinc (Zn) finger protein, interacts with the MRE transactivating the metallothionein gene [41]. Zn also binds to tubulin proteins stimulating MT assembly in vitro. Tubulins isolated from brains of Zn-deficient animals showed an impaired ability for polymerization of MTs [42]. As³⁺ upregulated methallothioneins possibly by its binding to sulfhydryls of tubulins or other proteins releasing bound Zn ions that in turn enhance MTF-1 affinity for MRE containing genes such as metallothionein [41]. The NF-E2-related factor-2 (Nrf2) regulates antioxidant response element (ARE)-mediated expression of γ-GCS heavy subunit and other xenobiotic metabolizing enzymes. Nrf2 binds to the cysteine-rich keap1 protein in the actin filaments as an inactive form in the cytoplasm. Upon oxidative stress such as As³⁺ exposure, modification of cysteine residues in the Keap1 protein results in the Nrf2 release and nuclear localization for ARE binding and transactivation of the genes [43]. Elevation of cellular metallothionein and γ-GCS/GSH may be a critical detoxification mechanism against As³⁺ toxicity.

Tubulin heterodimer binds two GTP molecules, one exchangeable in β-tubulin and another one nonexchangeale in α-tubulin. Upon polymerization, GTP bound to β tubulin is hydrolyzed to GDP by intrinsic tubulin GTPase activity. The resulting GDP-tubulin exhibits a reduced affinity for its neighbors and undergoes disassembly [35]. Thus, MTs exhibit dynamic equilibrium, growing and shrinking by the addition or loss of tubulin dimers from the ends of protofilaments. As³⁺ has been reported to inactivate the GTP binding site on tubulins, thus inhibiting the MT assembly in vitro [44]. The hydrolysis of GTP to GDP in β-tubulin increases the curvature of protofilaments, destabilizing MTs [45]. This suggests that modulating the curvature status of MTs can change its stability. Taxol binding to the β-tubulin subunit, specifically to peptides 1–31, 217–231 and 297–293 [46], has been shown to straighten the GDP protofilament and slow down the transition of protofilaments from straight to a curved configuration [45]. Taxol stabilization of MTs has been reported to resist depolymerization by Ca²⁺, cold temperature, dilution, etc. [47]. Here, we go on to demonstrate taxol antagonism on disassembly of MTs by sulfhydryl-directed agents such as As³⁺ and NEM. Presumably, As³⁺ binding to tubulin -SH-groups may enhance the curvature of protofilaments, thus instabilizing MTs.

In addition to the tubulin dimer, MTs also contain several minor proteins, i.e., MAPs. The procedures for metabolic labeling, extraction with detergent and Ca²⁺, analysis of MAPs and other related proteins on 2-D gels have been used in different cells in response to different agents [25,48]. As shown in our studies, As³⁺ induced disassembly of MTs. Autoradiograms of proteins present in the MT fraction under As³⁺ treatment conditions were compared with its “control” profile derived from cells without As³⁺ treatment to identify subtle changes in protein molecular species. A protein that is present in cells with intact MT but changed or absent when intact MT no longer exist in such cells (such as nocodazole and As³⁺ treatments) fulfilling the operational definition of MAPs. Because of its high resolution, the quantitative 2-D gel electrophoresis has been the most powerful technique used for identifying unknown MAP proteins. With this 2-D gel system, several MAP proteins have been identified [25,48]. However, only five of them were decreased by >50% in cells exposed to 10 μM As³⁺ [Figure 10(B)], indicating that not all MAPs were equally susceptible to this trivalent metalloid element. MAPs exist only in minute amounts in non-neuronal cells. Based on their apparent MW and pI on 2-D gels, none of these MAPs (Figure 10) appeared to resemble any existing MAP reported in the literature. This is not surprising since most MAPs identified so far, such as MAP1, MAP2, MAP3, Tau family and 210 kD protein (MAP4), etc., are from either the brain tissue or cells of neural origin [29,49,50,51]. Notably, several MAP, e.g., MAP-2 and Tau proteins isolated from the brain tissue, stimulate MT assembly and enhance MT stability both in vitro and in vivo [29,50]. Thus, results in giving for inhibition of MAPs by As³⁺ would lead to the enhancement of MT instability.

Immunohistochemical studies indicated severe loss of tubulin synthesis and distribution in As³⁺-dosed lungs [Figure 4(A2)]. Since absorbed As³⁺ is distributed throughout the body [5,6], all types of lung cells are targets for As³⁺ insult including interstitial fibroblasts. In view of MT functions, loss of MTs/tubulins, a cell structural protein, may be a critical basis for As-induced lung pathology such as bronchiectasis, bronchitis, COPD, etc. Synthesis of tubulin is subjected to a novel feedback autoregulation, i.e., the level of the free tubulin pool directly controls the level of tubulin expression [52]. Thus, As³⁺ disrupts MTs increasing the free tubulin pool, as a result, inhibiting new tubulin synthesis. Our studies have shown As³⁺ downregulation of tubulin synthesis occuring at the mRNA level, i.e., marked reduction of steady-state βI-tubulin expression in in vitro and in vivo models [Figure 3(B) and Figure 4(B)]. As reported, the binding of tubulin subunits to the nascent N-terminal tetrapeptide (Met-Arg-Glu-Ile) of β-tubulin triggers the adjacent ribosome to activate an RNase that degrades the polysome bound tubulin mRNA, inducing its destabilization [53]. In addition to its effects on tubulin mRNA stability, As³⁺ may also reduce tubulin mRNA initiation (synthesis) leading to low levels of steady-state tubulin mRNAs in lung cells and tissues.

MTs play a fundamental role in regulation of cell division. Mitosis requires precise execution of a number of MT-based processes such as reduplication and splitting of the centrosome, assembly of a bipolar spindle, attachment of spindle MTs to chromosomes through kinetochores, tension generation on kinetochores by the dynamics of bipolar spindle MTs, and finally pulling each chromatid to one of the spindle poles [54]. An error in any step of such interdependent subcellular processes could perturb chromosomal organization, resulting in failure of mitosis. In this study we have observed cell division aberrations in the forms of chromosome lagging, scattering and clustering associated with spindle MT injury in the cell model following As³⁺ exposure (Figure 2). These characteristic chromosomal disorientations are also evident in cells treated with colchicine, an MT disassembly reagent [55]. Disassembly of all spindle MTs by As³⁺ may induce chromosomal scattering and clustering at metaphase while partial injure of spindle MT (such as disconnection of a few spindle fibers with chromosomes and/or monopole or multipoles, etc.) may lead to chromosomal lagging at anaphase/telophase. The latter appears to result from escaping the mitotic block (i.e., passing through metaphase) as seen [Figure 2(i)]. Importantly, lagging chromosomes are believed to induce aneuploidy [56], which is in turn closely related to tumorigenesis [57]. Oxidation and methylation status of As displayed various effects on the mitotic apparatus [58]. As³⁺-resulted in production of aneuploid cells was associated with disruption of both the MT assembly and the spindle formation [59]. Thus, As³⁺-induced disassembly of spindle MTs and aberrations in the cell division have a critical biological significance for human tumor pathogenesis.

Briefly, present studies demonstrate two molecular targets of MTs for As³⁺ insult, i.e., tubulin -SH groups and MAPs. As^{3+ binding} to tubulin -SH groups and inhibition of MAPs enhanced MT instability leading to its disassembly. Increase in the free tubulin pool by As³⁺ as a result of MT disassembly triggered an autoregulation mechanism inducing inhibition of tubulin synthesis at protein and mRNA levels and thus further aggravating lung MT injury, which was accompanied by activation of cellular metallothionein and γ-GCS, a critical thiol defense mechanism. As³⁺ damage to MTs was antagonized by taxol. Cytoskeletal MT injury by As³⁺ may be a critical basis for pulmonary pathology. These findings are expected to provide a piece of information for preventing As³⁺ cellular toxicity in the lung.