ALTEX :: Alternatives to Animal Experiments

2001, VOLUME 2

Inter-Species Variations in Ochratoxin A Protein Binding and Uptake

Daniel R. Dietrich¹, Evelyn O´Brien¹, Alexandra Heussner¹, Michael Stock², Klaus Hochberg³ and Billy W. Day⁴

¹Environmental Toxicology, University of D-Konstanz; ²Food and Drug Administration, USA-Washington DC; ³Urology, Klinikum D-Konstanz, ⁴Department of Occupational Health, University of Pittsburgh, USA-Pittsburg, PA

SUMMARY

Chronic dietary intake of 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 be carcinogenic in rodents, with distinct species- and sex-differences in susceptibility. Despite several acute, sub-acute and chronic whole animal studies in diverse species, the basis for these sensitivity differences remain to be elucidated. Such differences could arise from variations in cellular OTA uptake and/or binding characteristics. Therefore, the aims of this study were to investigate these parameters in vitro using relevant tissue homogenates and cell culture models for comparison with in vivo and epidemiological observations. Protein binding studies were carried out using a classical competitive assay using ³H-OTA and homogenates from human, porcine, rat and mouse renal cortex tissue. Species-specific binding capacities were observed: human >> rat > pig > mouse. OTA bound human and porcine homogenates with higher affinity than rat and mouse with regard to competition with bromosulfophthalein (BSP). This binding could be inhibited in a concentration-dependent manner by piroxicam, indomethacin, nalidixic acid, ethacrynic acid, furosemide and probenecid, but not with fumonosin B1, para-aminohippurate (PAH) and several bile acids. ³H-OTA uptake was measured over 60 minutes in human (HKC) and porcine (PKC) primary renal epithelial cells and in the NRK52E and LLC-PK1 cell lines. Uptake over time remained relatively constant, however, distinct differences were observed in the amount of 3H-OTA taken up by the different cell types. Both HKC and PKC displayed a 10-15 fold higher uptake than that observed in LLCPK-1 and NRK-52E cells. The species-differences observed in this study reflect those observed by previous authors in whole animal studies and therefore, provide a good basis for further mechanistic investigations.