Abstract
Renal cell lines are frequently used models in toxicology. The aim of the experiments described here was to investigate the suitability of two of those renal cell lines, namely NRK-52E and LLC-PK1, as models for mid to late stage apoptosis under standard cell culture conditions; the latter means that testing was performed in the presence of serum in the culture media. Seven known inducers of apoptosis already positively tested by other investigators were chosen as test substances using chromatin condensation (Hoechst staining) as endpoint. These substances were cadmium chloride (CdCl2), dithiothreitol (DTT), sodium chloride (NaCl), mercuric chloride (HgCl2), tributyltin oxide (TBT-O), tributyltin chloride (TBT-Cl) and staurosporine. From these, only TBT-O, TBT-Cl and staurosporine induced morphological features typical of apoptosis in LLC-PK1 cells. Morphologically discerned apoptosis was confirmed by DNA fragmentation (DNA laddering assay) analysis. LLC-PK1 cells, but not NRK-52E cells, were shown to be suitable models of mid to late stage apoptosis under the conditions employed. TBT-O, TBT-Cl and staurosporine were shown to be suitable positive controls for apoptosis in renal cells in vitro.
Highlights
Continuous renal cell lines are frequently used cell models in toxicology due to their technical and economic advantages over primary cells or in vivo solutions
The aim of the experiments described here was to investigate the suitability of two of those renal cell lines, namely NRK-52E and LLC-PK1, as models for mid to late stage apoptosis under standard cell culture conditions; the latter means that testing was performed in the presence of serum in the culture media
Discerned apoptosis was confirmed by DNA fragmentation (DNA laddering assay) analysis
Summary
Continuous renal cell lines are frequently used cell models in toxicology due to their technical and economic advantages over primary cells or in vivo solutions. The aim of the project presented here was to investigate the suitability of two of these renal cell lines, namely NRK-52E and LLC-PK1, with respect to their sensitivity to various known apoptosis inducers using mid to late stage apoptosis, i.e. chromatin condensation and DNA fragmentation, as endpoints. Condensed chromatin with a distinct fragment size, roughly 180 - 200 base pairs and multiples thereof, is the result of a specific DNA fragmentation via cleavage by endogenous endonucleases. The latter DNA fragments can be used as a marker for apoptosis, e.g. via the DNA laddering assay [1]. Necrosis is typically characterized by random DNA fragmentation resulting in a DNA smear rather than a distinct ladder in DNA agarose gel electrophoresis
Sign-in/Register to view highlights & summary 
Published Version (
Free)
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call