Cytotoxicity, genotoxicity and apoptosis induced by ochratoxin A and citrinin in porcine kidney PK15 cells: Effects of single and combined mycotoxins

Abstract

The cytotoxic, genotoxic and apoptotic effects of ochratoxin A (OTA) and citrinin (CIT), and their combinations, were studied on porcine kidney PK15 cells. IC50 (24 h) of OTA and CIT measured with Trypan blue on cells exposed OTA (2-46 mM) and two CIT (10-130 mM) were 17.35+1.0 and 67.63+1.9 mM, respectively. Two OTA (6 and 10 mM) and two CIT (30 and 50 mM) doses as well as their combinations (6+30, 6+50, 10+30 and 10+50 mM) applied for 24 reduced cell viability to 86, 71, 86, 76, 68, 63, 64 and 43% of control, respectively. 48 h exposure to single mycotoxin further reduced cell viability to 76, 58, 76, 54%, respectively, while the combined exposure gave synergistic effect (reduction to 31, 20, 16 an 6 % respectively). Genotoxic and apoptotic effects of OTA and CIT, and their combinations were tested following 24 h exposure by scoring 2000 cells (DAPI) for the presence of micronuclei (MN), nuclear buds (NB), nucleoplasmic bridges (NB) and apoptotic cells (AC). Significant genotoxic and apoptotic effects were observed after exposure to 30 mM CIT (8.75%o MN, 7.25%o AC) and 50 mM CIT (16.75% MN, 8%o NB, 12.5%o AC) as compared to control (2.75%o MN, 3%o NB, 0.75%o AC). OTA (10 mM) significantly increased the frequency of MN (9.5%o ) and AC (5.75%o). Combined treatment synergistically increased AC (up to 87%o), NB (up to 26 %o) and NPB (up to 4%o), while MN frequency remained low probably because of decreased mitotic index.