Detection of cellular damage by hydrogen peroxide using SV40-T2 cells on shear horizontal surface acoustic wave (SH-SAW) sensor.

The rat lung epithelial cell line SV40-T2 was used to develop a cellular biosensing system to assay for environmental toxicants. The novel approach on which this system is based involves direct attachment of cultured rat or human cells onto a cell-adhesive matrix on the device through which shear horizontal surface acoustic waves (SH-SAW) are transmitted using 50 MHz SAW resonator. This novel design enables sensitive monitoring of changes of the electrophysical characteristics of cells, such as their conductivity and relative permittivity. A time-dependent change of phase of SAW and change of insertion loss (change of amplitude) were observed when the cells were treated with 0.5 or 1.0 mM H2O2. The change of insertion loss was biphasic, with an early phase (1-3 h) and a late phase (3-6 h). The late phase coincided with the destruction of cell-cell tight junctions detected by measurement of the transepithelial electrical resistance and paracellular permeability; in contrast, the early phase coincided with the destruction of intracellular actin filaments by H2O2. The early-phase effect of H2O2 on phase shift may be attributable to the change of intracellular permittivity by a change of cellular polarity. Immunofluorescence microscopy showed the disappearance of zonula occludens protein 1 from the region of cell-cell contact. These results suggest the correlation between the change of insertion loss as an SAW parameter and the destruction of tight junctions of the cells on the SH-SAW device in the late phase.

Correlation between the destruction of tight junction by patulin treatment and increase of phosphorylation of ZO-1 in Caco-2 human colon cancer cells.

Patulin is a mycotoxin and its contamination of food has been reported to cause gastrointestinal inflammation, ulcers, and bleeding. The toxicity of patulin is thought to be due to the destruction of tight junctions (TJs) in gastrointestinal tissues. However, the precise mechanism has not been clarified. Here, we investigated the phosphorylation of TJ components. The transepithelial electrical resistance (TER) of Caco-2 human colon cancer cells decreased gradually during the first 24h of treatment with 50µM patulin. Immunofluorescence microscopy showed that the TJ proteins ZO-1 and claudin-4, but not occludin, had decreased after 24h and decreased from the cell-cell contact regions of TJs after 48h of patulin treatment. Western blotting showed that the level of ZO-1 decreased after 48h of patulin treatment, but the levels of claudin-4 and occludin remained at the initial level until 72h. Phosphorylation of ZO-1 was detected by 24h and increased markedly after 72h of patulin treatment. However, phosphorylation of claudin-4 and occludin was not detected by probing with anti-phosphotyrosine antibody. Immunoprecipitation showed that interaction of ZO-1 with claudin-4 had decreased after 48h and was completely absent after 72h. These results suggest that phosphorylation caused the degradation of ZO-1 protein and the decrease in TER induced by patulin treatment of Caco-2 cells.