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, 175 (5), 2171-83

Nano-scaled Particles of Titanium Dioxide Convert Benign Mouse Fibrosarcoma Cells Into Aggressive Tumor Cells

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Nano-scaled Particles of Titanium Dioxide Convert Benign Mouse Fibrosarcoma Cells Into Aggressive Tumor Cells

Kunishige Onuma et al. Am J Pathol.

Abstract

Nanoparticles are prevalent in both commercial and medicinal products; however, the contribution of nanomaterials to carcinogenesis remains unclear. We therefore examined the effects of nano-sized titanium dioxide (TiO(2)) on poorly tumorigenic and nonmetastatic QR-32 fibrosarcoma cells. We found that mice that were cotransplanted subcutaneously with QR-32 cells and nano-sized TiO(2), either uncoated (TiO(2)-1, hydrophilic) or coated with stearic acid (TiO(2)-2, hydrophobic), did not form tumors. However, QR-32 cells became tumorigenic after injection into sites previously implanted with TiO(2)-1, but not TiO(2)-2, and these developing tumors acquired metastatic phenotypes. No differences were observed either histologically or in inflammatory cytokine mRNA expression between TiO(2)-1 and TiO(2)-2 treatments. However, TiO(2)-2, but not TiO(2)-1, generated high levels of reactive oxygen species (ROS) in cell-free conditions. Although both TiO(2)-1 and TiO(2)-2 resulted in intracellular ROS formation, TiO(2)-2 elicited a stronger response, resulting in cytotoxicity to the QR-32 cells. Moreover, TiO(2)-2, but not TiO(2)-1, led to the development of nuclear interstices and multinucleate cells. Cells that survived the TiO(2) toxicity acquired a tumorigenic phenotype. TiO(2)-induced ROS formation and its related cell injury were inhibited by the addition of antioxidant N-acetyl-l-cysteine. These results indicate that nano-sized TiO(2) has the potential to convert benign tumor cells into malignant ones through the generation of ROS in the target cells.

Figures

Figure 1
Figure 1
Thymosin ß4 gene expression in QRnP tumor cells and detection of 8-OHdG and HNE in titanium dioxide nanoparticle implantation sites. A: Tumor cell lines were established from tumors derived from QR-32 cells that had been injected into the pre-implantation site of either TiO2−1 (QRnP-1 to 7) or TiO2−2 (QRnP-8) implanted mice. The Figure shows RT-PCR analysis for thymosin ß4 and GAPDH gene expression. B: Five mg of TiO2−1 or TiO2−2 were implanted subcutaneously in mice. Large (black arrowhead) and small (white arrowheads) deposits of the nanoparticles are indicated. Tissues were removed on the indicated days and stained by the Azan method or subjected to immunohistochemical study for detection of 8-hydroxy-2′-deoxyguanosine (8-OHdG) and 4-hydroxy-2-nonenal (HNE). Scale bar: 200 μm.
Figure 2
Figure 2
Quantitative real-time PCR for inflammation-related cytokines and growth factors. Real-time RT-PCR analysis was performed to quantify changes in mRNA expression of eight cytokine/growth factors in the TiO2−1- or TiO2−2- implanted tissues. *P < 0.05 and **P < 0.01 vs. TiO2−1.
Figure 3
Figure 3
Formation of reactive oxygen species and mediation of cytotoxicity to QR-32 cells by titanium dioxide nanoparticles. A: Generation of reactive oxygen species (ROS) by nano-TiO2 in a cell-free system was determined using the 2′, 7′-dichlorodihydrofluorescein diacetate reagent. *P < 0.001 vs. individual nano-TiO2 treatment. **P < 0.05 and ***P < 0.001 vs. medium alone. B: Intracellular ROS generation in QR-32 cells co-cultured with 312 μg/ml of nano-TiO2 was determined using cell-permeable 5-(and-6)-chloromethyl-2′, 7′-dichlorodihydrofluorescein diacetate acetyl ester. *P < 0.05 and **P < 0.001 vs. QR-32 cells. C: Production of nitric oxide (NO) was determined by measuring both nitrite (NO2) and nitrate (NO3) concentrations in the culture medium. *P < 0.05 and **P < 0.01 vs. QR-32 cell alone. Peritoneal exudate cells (PEC) were collected from murine peritoneal cavities 5 days after implantation of a piece of gelatin sponge and used as positive controls for NO production. D: Treatment of adherent (from 1 hour to 48 hours) or suspended (for 72 hours; 72S) QR-32 cells with nano-TiO2 (312 μg/ml). After exposure to the nano-TiO2, the cells were harvested and counted. *P < 0.05 and **P < 0.001 vs. QR-32 cells. Closed bars: TiO2−1 alone (A) or QR-32 cells treated with TiO2−1 (B, C, D); open bars: TiO2−2 alone (A) or QR-32 cells treated with TiO2−2 (B, C, D); dotted bars: culture medium alone (A) or non-treated QR-32 cells (B, C, D). N-acetyl-l-cysteine (NAC) and aminoguanidine (AG) were used as inhibitors of ROS and NO, respectively.
Figure 4
Figure 4
Transmission electron microscopic examination of QR-32 cells after coculture with nano-TiO2. Transmission electron microscopy revealed that hydrophilic nano-TiO2 (TiO2−1; A, C, E) was incorporated into enlarged follicle-like organelles in the cytoplasm of QR-32 cells. Hydrophobic nano-TiO2 (TiO2−2; B, D, F) formed interstices around the nuclear membrane and caused a multinucleated phenotype in QR-32 cells. Spontaneous aggregation of nano-TiO2 was seen in the extracellular space. Original magnification: ×3000 (A); ×3000 (B); ×10,000 (C); ×7000 (D); ×15,000 (E), and ×20,000 (F). Scale bars= 5 μm (A); 5 μm (B); 2 μm (C); 5 μm (D); 1 μm (E); and 1 μm (F).

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