A potential role of calcium in apoptosis and aberrant chromatin forms in porcine kidney PK15 cells induced by individual and combined ochratoxin A and citrinin
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The aim of this study was to establish the involvement of calcium signalling in genotoxicity, apoptosis and necrosis evoked by ochratoxin A (OTA) and citrinin (CTN) alone or in combination in porcine kidney PK15 cells. Cell proliferation test (MTT) and trypan blue assays (24 h) demonstrated that CTN (IC50 = 73.5 ± 1.0, 75.4 ± 1.4 μM, respectively) was less toxic than OTA (IC50 = 14.0 ± 2.4, 20.5 ± 1.0 μM, respectively). To test their cytotoxic interactions, two doses of single OTA (6 and 10 μM) and CTN (30 and 50 μM) and their combinations were applied. Combined treatment showed additive cytotoxic effects. OTA and CTN induced dose-dependent increase in cytosolic calcium level (assessed with Fura-2 AM). However, combined treatment did not provoke additional increase in calcium signal. The rate of apoptosis and necrosis (DAPI-antifade staining) was significantly higher after 12 h than 24 h, while the frequencies of micronuclei (MNs) and nuclear buds (NBs) were higher after 24 h than 12 h treatment. Combined exposure resulted in apoptotic and necrotic synergism, while genotoxic effects of OTA + CTN were noted as antagonistic or additive. Co-exposure of cells to calcium chelator BAPTA-AM significantly reduced CTN and OTA + CTN-evoked apoptosis. Twenty-four hour after co-exposure to BAPTA-AM and a single OTA and CTN, MNs significantly decreased while NBs dropped significantly after co-treatment with BAPTA-AM and OTA + CTN. In conclusion, disturbance of Ca2+ homeostasis caused by OTA and CTN plays a significant role in cell genotoxicity and death.
KeywordsNephrotoxins Genotoxicity Necrosis BAPTA-AM Mycotoxin synergism
We wish to thank Professor Josip Madić, PhD, and Snjezana Kovač, MSc (Veterinary Faculty, University of Zagreb, Croatia), for providing the PK15 cells. Dr Andrey Y. Abramov’s help in calcium measurements is greatly acknowledged. This work was financially supported by the Ministry of Science, Education and Sports of the Republic of Croatia (Grants No. 022-0222148-2137, 022-0222148-2142, 006-0061117-1242). Ana-Marija Domijan’s stay in Department of Molecular Neuroscience, UCL Institute of Neurology was financially supported by The National Foundation for Science, Higher Education and Technological Development of the Republic of Croatia.
Conflict of interest
The authors declare that there are no conflicts of interest.
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