Induction of micronuclei by ochratoxin A is a sensitive parameter of its genotoxicity in cultured cells

Abstract

In order to better characterize the ochratoxin A (OTA)-induced DNA damage and to further investigate factors which may modulate dose-effect relationships in cells, the induction of micronuclei was studied in V79 Chinese hamster fibroblast cells and in primary cultures of porcine urothelial bladder epithelial cells (PUBEC). OTA was able to induce micronuclei in PUBEC and V79 cells at concentrations below those which were overtly cytotoxic. OTA concentrations between 0.03 and 1 μM caused a dose-dependent increase of micronuclei in V79 cells (up to 3-fold compared to controls); but the lowest tested concentration of 0.01 μM OTA did not induce a higher frequency of micronuclei than in the solvent control, indicative of an apparent threshold. Clear evidence for genotoxic effects was also found in PUBEC cultures treated with OTA concentrations of 1 μM and more, although the dose-effect relationship in PUBEC was more variable for several freshly isolated cell batches, pointing to differences in susceptibility to OTA between bladder cells from different donor animals. The chromosomal genotoxicity of OTA demonstrated in this study is in general accord with previous findings on the induction of clastogenic effects and oxidative DNA damage by OTA. In both cases, the shape of the dose-response curve at very low OTA concentrations supports the existence of a threshold for its genotoxicity.

The emerging Fusarium toxin enniatin B: in-vitro studies on its genotoxic potential and cytotoxicity in V79 cells in relation to other mycotoxins

“…By means of a battery of short-term tests, used in our lab also for other compounds (Föllmann and Lucas 2003; a In a few cases the curves for enniatin B showed a hump at higher levels, which led to less reliable estimates of the IC 50 value (up to 53 μM); these data are not inlucded here b Cytotoxic potency of ochratoxin A is known to be strongly dependent upon serum content of the treatment medium, due to its high binding to serum proteins (Degen et al 1995;Stock 2004) Bonacker et al 2004;Dorn et al 2008;Föllmann et al 2007), we have generated a profile of the genotoxicity of enniatin B. With regard to the choice of in-vitro assays, it may suffice to say that tests with bacteria and mammalian cells, such as the Ames assay and the HPRT assay, are considered the crucial basis for detecting different types of mutations.…”

“…The micronucleus assay with V79 cells was carried out as described before (Bonacker et al 2004;Dorn et al 2008;Föllmann et al 2007), with minor adaptations. Cells were treated for 18 h with enniatin B (0.03-10 μM in medium); vincristin and methylmethane sulfonate served as positivecontrol agents for aneugenic and clastogenic effects, respectively.…”

Cytotoxic Potency of Mycotoxins in Cultures of V79 Lung Fibroblast Cells

“…The concentration of NR dye, extracted from V79 cells and measured by absorption spectrometry, is closely correlated to the number of vital cells. The assay is thus well suited to determine the cytoxicity of various agents including mycotoxins Föllmann et al 2007;Bonacker et al 2004;Dorn et al 2007). Briefly, 5000 V79 cells were seeded in 200 µl culture medium per well, in transparent 96-well microtiter plates, and cultured for 24 h. Medium was then withdrawn and cells were treated for 48 h with graded concentrations of mycotoxins in culture medium with 5% FCS; the final solvent concentration did not exceed 0.1% (v/v) in the mycotoxin-treated cultures and the concomitant solvent controls.…”

Synergism of aromatic amines and benzo[a]pyrene in induction of Ah receptor-dependent genes

“…PUBEC were seeded in 96-well microtiter plates in serum-free medium, and evaluated as described previously (Föllmann et al 2000b(Föllmann et al , 2007Wolf et al 2005). In brief, 20,000 PUBEC in 200 l culture medium were seeded per well in 96-well culture plates.…”