Osteopontin mRNA expression by rat mesothelial cells exposed to multi-walled carbon nanotubes as a potential biomarker of chronic neoplastic transformation in vitro

Abstract

Mesothelioma is a cancer of the lung pleura primarily associated with inhalation of asbestos fibers. Multi-walled carbon nanotubes (MWCNTs) are engineered nanomaterials that pose a potential risk for mesothelioma due to properties that are similar to asbestos. Inhaled MWCNTs migrate to the pleura in rodents and some types cause mesothelioma. Like asbestos, there is a diversity of MWCNT types. We investigated the neoplastic potential of tangled (tMWCNT) versus rigid (rMWCNT) after chronic exposure using serial passages of rat mesothelial cells in vitro. Normal rat mesothelial (NRM2) cells were exposed to tMWCNTs or rMWCNTs for 45 weeks over 85 passages to determine if exposure resulted in transformation to a neoplastic phenotype. Rat mesothelioma (ME1) cells were used as a positive control. Osteopontin (OPN) mRNA was assayed as a biomarker of transformation by real time quantitative polymerase chain reaction (qPCR) and transformation was determined by a cell invasion assay. Exposure to rMWCNTs, but not tMWCNTs, resulted in transformation of NRM2 cells into an invasive phenotype that was similar to ME1 cells. Moreover, exposure of NRM2 cells to rMWCNTs increased OPN mRNA that correlated with cellular transformation. These data suggest that OPN is a potential biomarker that should be further investigated to screen the carcinogenicity of MWCNTs in vitro.

Keywords: Carbon nanotubes; Mesothelial cells; Mesothelioma; Osteopontin.

Fig. 1.

Transmission electron microscope images of nanotubes used in our in vitro experiments. A) tangled (tMWCNT). B) rigid (rMWCNT).

Fig. 2.

Light microscopy images of NRM2 and ME1 cells at low passages (P12–13) and high passage (P80) with or without exposure to tMWCNTs or rMWCNTs.

Fig. 3.

OPN mRNA expression and cell invasion in NRM2 (P14, P12) & ME1 (P15, P13) cells as indicators of mesothelioma. A) OPN mRNA measured by real time qPCR. B) Quantification of cell invasion across an extracellular matrix after 24hrs. *** p < 0.001, ** p < 0.01 compared to control. Data are from a single experiment representative of three independent experiments.

Fig. 4.

Osteopontin mRNA expression with exposure to tMWCNTs or rMWCNTs with increasing cell passages. ***p < 0.001, *p < 0.05 compared to passage # control. ^###p < 0.001, ^##p < 0.001 between tMWCNT and rMWCNT. Data are from a single experiment representative of three independent experiments.

Fig. 5.

Cell invasion assay with high passage NRM2 cells exposed to tMWCNTs or rMWCNTs. Low passage (P12) as negative control. *** p < 0.001, ** p < 0.01 compared to P85 control. ^###p < 0.001 between P85 tMWCNT and P85 rMWCNT. Data are from a single experiment representative of three experiments. Each experiment contained 16 replicates per group.