Induction of micronuclei with ochratoxin A in ovine seminal vesicle cell cultures.

Abstract

The genotoxic potential of the carcinogenic mycotoxin of ochratoxin A (OTA) has been investigated by means of an in vitro micronucleus assay, an endpoint for genotoxicity which has not been studied previously for OTA. OTA was found to induce dose-dependently micronuclei (MN) in cytokinesis-blocked binucleated ovine seminal vesicle (OSV) cell cultures, which had been treated with mycotoxin (12-30 microM) for 6 h in medium containing 10% fetal calf serum. For comparison, OSV cells were treated with colcemid (0.02-0.06 micrograms/ml), or 4-nitroquinoline N-oxide (NQO; 0.5 microM), a typical aneugen and clastogen, respectively. All test compounds increased the frequency of MN in OSV cells, the highest level being induced by 10 microM OTA. When MN were characterized by indirect immunofluorescence microscopy using anti-kinetochore (CREST) antibodies, the majority of MN in colcemid-treated cells was CREST-reactive (> 70% kinetochore positive); as expected, this fraction was < 10% for the NQO-treatment group. In cells treated with OTA the fraction of kinetochore positive MN was similar (33-40%) to that observed in solvent controls (38%). These data indicate that OTA induces MN apparently by a mixed, although predominantly clastogenic mode of action. OSV cells lack monooxygenase activity but express high prostaglandin H synthase (PGHS) activity. When cells were treated with OTA in the presence of indomethacin (10 and 10 microM), a well known inhibitor of PGHS, the frequency of MN induced by OTA was not decreased, but rather increased. This indicates that metabolic activation of OTA by PGHS seems not to be required for genotoxicity. The increased MN induction in OSV cell cultures is most likely due to competition in indomethacin with OTA for binding to serum proteins thus raising the fraction of free mycotoxin.