**3. Transient and high-dose exposure to asbestos fibers in MT-2 cells: Cytotoxicity**

The left side of **Figure 1** shows findings concerning the transient and high-dose exposure to asbestos fibers in MT-2 cells. The cells proceed to apoptosis just as alveolar epithelial cells and pleural mesothelial cells were previously reported [14–17].

Asbestos exposure caused production of ROS. **Figure 1** shows the production of superoxide anion (O<sup>2</sup> − ) as positive for hydroethidine analyzed by flow cytometry. Proapoptotic signaling molecules in the mitogen-activated protein kinase (MAPK) pathway such as JNK and p38 were then phosphorylated. Cytochrome c in mitochondria was then released into the cytoplasm. As a result, the proapoptotic molecule BAX was upregulated in the cells. These findings indicated that the mitochondrial apoptotic pathway was activated by asbestos exposure. Caspase 9 and 3 were then truncated into active forms to cause apoptosis of cells [31, 32].

In addition, cellular phenomena such as growth inhibition, appearance of apoptosis analyzed by annexin V staining (as an early event), activation of caspase 3, positivity of Tunel staining (a late event), and ROS production were compared between MT-2 cells exposed to fibers of chrysotile and crocidolite (CH and CR, respectively in **Figure 1**). Since CR contains a massive level of iron compared with CH, ROS production was higher in MT-2 cells exposed to CR. However, other events (the degree of growth inhibition, appearance of apoptosis assayed by different methods) were stronger in MT-2 cells exposed to CH compared to CR, although these were just comparisons between these two fibers and apoptosis was certainly caused by asbestos exposure on MT-2 cells [31–33].

Taken together, the cytotoxicity of asbestos fibers on human T cells was caused by a mechanism similar to that demonstrated for alveolar epithelial and pleural mesothelial cells.

Cytotoxicity Caused by Asbestos Fibers and Acquisition of Resistance by Continuous Exposure…

http://dx.doi.org/10.5772/intechopen.72064

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The initial aim of asbestos exposure on the MT-2 cell line was to establish a model of continuous, recurrent, and relatively low-dose exposure on human T cells and observe which kind of alterations occur under conditions of continuous and low-dose exposure as found with

Exposed doses for continuous exposure were then determined as doses which caused apoptosis in less than half of MT-2 cells. Doses included 5 or 10 μg/ml in culture flasks, as shown on the left side of **Figure 1**. A dose of 10 μg/ml of chrysotile caused various apoptotic cellular events, although the degrees of these events were less than those resulting from exposure to 25 μg/ml of chrysotile [31, 34]. Since MT-2 cells were derived from T cells, they were grown by floating in the culture. Thus, the concentration of asbestos fibers was determined using μg/ml, although various adherent cells such as epithelial and mesothelial cells were

ture twice a week and substitute asbestos fibers according to the determined concentrations. To monitor cellular alteration, the asbestos fibers were removed from continuous culture by density gradient centrifugation using LymphoPrep (gradient = 1.077) and continuously exposed cells were analyzed for the occurrence of apoptosis after transient exposure to highdose asbestos fibers (which caused apoptosis in most of the MT-2 cells such as 25–50 μg/ml) [31, 32, 34]. After 8 months of continuous exposure, the appearance of apoptosis was reduced in the continuously exposed subline. This acquisition of resistance to asbestos-induced apoptosis was sustained in long-term cultures (until now, the sublines were cultured with asbes-

After 1 year of continuous exposure, the cellular features of the continuously exposed subline (MT-2 CE) were compared to those of the original MT-2 cells (MT-2Org, which were never exposed to asbestos fibers) as shown on the right side of **Figure 1**. A consideration of cytokine production showed that there was an excess production of interleukin (IL)-10 in MT-2 CE relative to MT-2Org. The regulation of IL-10 production was mediated by Src kinase, since PPT, the Src inhibitor, reduced IL-10 production in both MT-2Org and MT-2 CE cells. The excessively produced IL-10 was then utilized by the autocrine mechanism, since MT-2 possesses the IL-10 receptor (R) at its surface. As a result of IL-10 utilization, the signaling molecule, signal transducers, and the activator of transcription (STAT)-3, located downstream of IL-10R, were phosphorylated to a higher level in MT-2 CE compared to MT-2Org. Since the antiapoptotic molecule Bcl-2 is located downstream of STAT3, the expression of Bcl2 was upregulated in MT-2 CE compared to that in MT-2Org. The Bax/Bcl2 ratio was lower in MT-2 CE than in MT-2Org, as shown on the right side of **Figure 1**. In order to investigate the importance of Bcl-2 in MT-2 CE concerning acquisition of resistance to asbestos-induced apoptosis, siRNA for Bcl-2 was introduced into MT-2 CE cells and the occurrence of apoptosis and growth inhibition following transient and high-dose exposure to asbestos fibers were examined. As

in the culture. The MT-2 cell culture was continued with a subcul-

**4. Continuous low-dose exposure to asbestos fibers in MT-2 cells:** 

human populations exposed to asbestos, such as the occurrence of cancers.

**Resistance to cytotoxicity**

measured using μg/cm<sup>2</sup>

tos fibers) [31, 34].

**Figure 1.** Schematic representation of the effects of the asbestos fibers chrysotile (CH) and crocidolite (CR) on MT-2 cells, a human T cell leukemia virus (HTLV)-1 immortalized human polyclonal T cell line [44–46]. Left side: Cellular and molecular alterations in MT-2 cells following transient and relatively high-dose exposure to CH or CR are summarized [32, 33]. Cells produced O<sup>2</sup> − , proapoptotic signaling molecules such as JNK and p38 were phosphorylated, cytochrome c was released from mitochondria to the cytoplasm, and caspases 9 and 3 were truncated into active forms. Cells then proceeded to apoptosis. A comparison of the effects caused by CH or CR showed that reactive oxygen species (ROS) production was greater with CR exposure, whereas growth inhibition and the level of apoptosis were greater following CH exposure. Right side: The effects of continuous and relatively low-dose exposure are summarized. MT-2 CE (a continuously exposed subline) revealed excess IL-10 production via Src kinase and phosphorylation of STAT3 resulting in upregulation of Bcl-2 [34]. In addition, the transcription factor FoxO1 was reduced in MT-2 CE, causing a reduction of proapoptotic molecules such as puma, bim, and FasL [36]. Both exposures contributed to the development of resistance to asbestos-induced apoptosis in MT-2 CE cells [44–46].

Taken together, the cytotoxicity of asbestos fibers on human T cells was caused by a mechanism similar to that demonstrated for alveolar epithelial and pleural mesothelial cells.
