ALTEX :: Alternatives to Animal Experiments

2001, VOLUME 2

Antiproliferative and Cell Cycle Specific Effects of Ochratoxin A in LLCPK-1, NRK-52E and Porcine Primary Proximal Kidney Cells

Stefanie B. Dreger, Evelyn O'Brien and Daniel R. Dietrich

Environmental Toxicology, University of D-Konstanz

SUMMARY

Ochratoxin A (OTA) is a mycotoxin produced as a secondary metabolite by certain Aspergillus and Penicillium species. It is commonly found as a contaminant of both human and animal foodstuffs. All human blood samples tested to date have proved positive for OTA. Average daily intake in humans is estimated to be approximately 5-10 ng/kg. OTA has been demonstrated to induce nephropathy as well as to be immunotoxic in pigs (antiproliferative effect in T-cells), teratogenic in several species and to cause renal tumours in rodents following chronic dietary intake. A disruption in the normal cellular proliferation control could contribute to the observed induction of nephropathy, immunotoxicity and teratogenicity of OTA as well as to its carcinogenic effects. Therefore, we investigated the effect of OTA on proliferation rates of LLCPK-1, NRK-52E and porcine primary proximal kidney cells (PKC) following acute exposure. Cells were exposed to increasing concentrations of OTA over 24, 48 and 72 hOUrs. Antiproliferative effects were determined using a Coulter counter. The concentration producing a 50% proliferative arrest (GI50) in LLCPK-1 and NRK-53E cells lines was 21 µM, with PKCs displaying a GI50 of 15 µM. Standard flow cytometric analysis of propidium iodide stained DNA was used to investigate the nature of this growth inhibitory effect. OTA was found to increase the percentage of cells residing in the G2/M phases of the cell cycle. More detailed analysis, (fluorescence-microscopic mitotic index of Hoechst-stained DNA) demonstrated no increased numbers of cells in M-phase thus suggesting an OTA mediated specific blockade of the cell cycle located to the G2 phase in all three cell types. As the G2 phase of the cell cycle is where proof-reading of replicated DNA and intracellular protein expression (cyclins, cdks) occur before entry into mitosis, an increased residency of cells in this cell cycle phase could indicated dysregulated proliferative control which could be a factor in disease development.