ALTEX :: Alternatives to Animal Experiments

2001, VOLUME 2

Comparison of Cytotoxic Effects of Ochratoxin A and B on Human, Rat and Porcine Renal Cells

Alexandra Heussner1, Michael Stack2, Klaus Hochberg3 and Daniel R. Dietrich1

1Environmental Toxicology, University of D-Konstanz; 2Food and Drug Administration, USA-Washington D.C; 3Urology, Klinikum Konstanz, D-Konstanz

SUMMARY

Chronic ingestion of the ubiquitous mycotoxin ochratoxin A (OTA) via contaminated food (bread, beer, coffee, wine, meat) has been associated with increased incidences of urothelial tumors and nephropathy in humans (e.g. Balkan Endemic Nephropathy, BEN). It has also been demonstrated to cause nephropathies in pigs and to be carcinogenic in rodents. Susceptibility to OTA toxicity displays large species- and sex-differences, the cause(s) of which are unknown but could relate to differences in cytotoxic responses. This study investigated the cytotoxic effects of OTA and its less toxic structural analogue OTB on primary renal cortex cells from humans (biopsy material), both sexes of F344 rats, and from improved German hybrid pigs and a porcine renal cell line, LLC-PK1, in an attempt to characterise the observed in vivo differences using in vitro test systems.Cells were exposed to OTA and OTB at concentrations ranging from 1 nM to 100 µM for 24 to 96 hours. Following incubation several standard cytotoxicity endpoints were assessed: Neutral red uptake, MTT reduction and cell number analyses (Coulter counter) to show differences in proliferation. Supravital fluorescent staining techniques as well as electron microscopy were employed to detect toxin-induced morphological changes. A range of fluorescent labeled antibodies to cytoskeletal proteins (e.g. vimentin, cytokeratins, actin, talin, tubulin) were used to demonstrate OTA- and OTB-induced morphological changes. Although all cells showed similar toxin-induced alterations, distinct species-differences were observed with respect to their relative sensitivities: porcine kidney cells > human kidney cells > rat kidney cells/LLCPK-1. Therefore some of the observed species-differences in sensitivity to OTA (and OTB) toxicity in vivo can be demonstrated using in vitro models.