In vitro studies to evaluate apoptosis induction by very low doses of ochratoxin a in rabbit splenocytes and peripheral blood mononuclear cells

2006 
Ochratoxin A (OTA), found commonly as a natural contaminant of food and feedstuffs, is a potent nephrotoxin. OTA has been shown to be immunosuppressive both in vivo and in vitro in many species. Apoptosis of immune cells has recently been proposed as a possible mechanism leading to immunosuppression caused by several agents. The present study was designed to evaluate the ability of OTA, at very low concentrations, in vitro, to induce apoptotic changes in splenocytes and peripheral blood mononuclear cells (PBMCs) of rabbits up to 48 hours of exposure. Pooled splenocytes and PBMCs obtained from three young New Zealand White rabbits, aged about 2 months, were cultured in vitro and exposed to 0.5μ M, 1μ M and 5μ M of OTA for 12, 24, 36 and 48 hours. Presence or absence of apoptosis was determined by the quantification of DNA hypodiploidy by flow cytometry, microscopic examination of acridine orange stained nuclei, and analysis of extracted DNA fragments. The proportion of splenocytes in the pre-G1 phase ranged from 9.47 to 12.06% and 8.37 to 12.31% for the control and OTA treated groups, respectively. Similarly, the proportion of pre-G1 phase of cell cycle ranged from 12.42 to 19.77% and 13.66 to 21.08% for the control and OTA treated PBMCs. The apoptotic index obtained through counting of acridine orange stained nuclei ranged from 10.5 to 21% for the controls and 13.5 to 22% for the OTA treated cells. Typical ladder pattern as expected for apoptotic changes was not observed in the DNA extracted from the OTA-treated splenocytes and PBMCs. Statistical analysis showed that there was no significant difference (p > 0.05) between the controls and the OTA treated cells in induction of apoptosis, as analyzed by the three methods. It was concluded from the present study that a very low dose of OTA ranging from 0.5 to 5μ M was not able to induce apoptosis in vitro in rabbit splenocytes and PBMCs.
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