Sensitive endpoints for evaluating cadmium-induced acute toxicity in LLC-PK1 cells.
Cadmium chloride (CdCl(2)) is a nephrotoxicant that causes damage to the proximal tubular epithelium. In vivo, it increases the permeability of epithelial surfaces, while in vitro, it acts on active trans-epithelial ion transport. The purpose of this study was to investigate CdCl(2) effects on a porcine renal proximal tubular epithelial cell line (LLC-PK1), and, in particular, to identify sensitive endpoints revealing damage both at the epithelial barrier level and at the molecular level. After exposure of the cells to CdCl(2), trans-epithelial resistance (TER) decreased while paracellular permeability (PCP) increased, indicating a structural alteration of the junctional complex. At the molecular level, we observed an increase in protective proteins, such as metallothioneins (MTs) and heat shock proteins (HSP70), starting from 25 microM CdCl(2), together with alterations in cytoskeleton organization. Production of reactive oxygen species (ROS) was also evident, indicating cellular oxidative stress. Our data indicate that CdCl(2) toxicity can be detected at the barrier level and at the molecular level at low concentrations, at which cytotoxicity assays are unable to show any damage. Therefore, these endpoints should prove very useful in studying heavy metal-induced acute toxicity. Exposure of the cells to higher concentrations of CdCl(2) (50 microM) revealed the initiation of apoptosis, mediated by caspase-3.
Gennari
Alessandra
A
ECVAM, Institute for Health and Consumer Protection, Joint Research Centre, European Commission, Via Fermi, 1, 21020 Ispra (Va), Italy. alessandra.gennari@jrc.it
Cortese
Elena
E
Boveri
Monica
M
Casado
Juan
J
Prieto
Pilar
P
D000208:Acute Disease; D000818:Animals; D017209:Apoptosis / Q000187:drug effects; D001692:Biological Transport / Q000187:drug effects; D015153:Blotting, Western; D019256:Cadmium Chloride / Q000633:toxicity*; D002105:Cadmium Poisoning / Q000378:metabolism* / Q000473:pathology; D002470:Cell Survival / Q000187:drug effects; D004847:Epithelial Cells; D005434:Flow Cytometry; D018840:HSP70 Heat-Shock Proteins / Q000378:metabolism; D007150:Immunohistochemistry; D007674:Kidney Diseases / Q000139:chemically induced* / Q000378:metabolism / Q000473:pathology; D007687:Kidney Tubules, Proximal / Q000187:drug effects* / Q000378:metabolism / Q000473:pathology; D007770:L-Lactate Dehydrogenase / Q000378:metabolism; D018374:LLC-PK1 Cells; D008668:Metallothionein / Q000378:metabolism; D017382:Reactive Oxygen Species / Q000378:metabolism; D013552:Swine
D018840:HSP70 Heat-Shock Proteins; D017382:Reactive Oxygen Species; D008668:Metallothionein; D007770:L-Lactate Dehydrogenase; D019256:Cadmium Chloride