Abstract
In the present study, we investigated the induction of apoptosis by N-nitrosopyrrolidine (NPYR) and N-nitrosodimethy-lamine (NDMA) in two human cell lines: HL-60 (leukemia) and HepG2 (hepatoma). Apoptotic cells were identified by: 1) chromatin condensation, 2) flow cytometry analysis and 3) poly (ADP-ribose) polymerase cleavage. Both cell lines exhibited morphological changes consistent with apoptotic events following treatment with N-nitrosamines. Flow cytometry analysis showed that both N-nitrosamines induced apoptotic cell death in a concentration and time dependent- manner. NPYR was stronger than NDMA, since it induced a significant apoptotic cell death after 72 h starting from a concentration of 10 mM, whereas NDMA was effective at 27 mM. Furthermore, NPYR and NDMA caused the cleavage of PARP in HL-60 cells whereas no PARP cleavage was detected in HepG2 cells. However, NPYR- and NDMA-induced cell death in HepG2 cells was prevented by specific caspase inhibitors. Caspase-8 mediated main pathway and was responsible for 76% (NPYR) and 64% (NDMA) inhibition of apoptosis. The data demonstrate that NPYR and NDMA induce apoptosis in HL-60 and HepG2 cell lines via caspase-dependent pathway.
Highlights
The N-nitroso compounds (NNC) are recognized as one of the most potent chemical mutagens and carcinogens present in environment and in food [1]
We investigated the induction of apoptosis by N-nitrosopyrrolidine (NPYR) and N-nitrosodimethylamine (NDMA) in two human cell lines: HL-60 and HepG2
Cells were treated with 10-50 mM NPYR or 27-135 mM NDMA for different time periods (24-72 h), and nuclear morphology was observed by fluorescence microscopy using acridine orange. 24 hours treatment of HL-60 cells at the highest doses of NPYR (50 mM) and NDMA (135 mM) induced 58% and 39% of apoptosis, respectively
Summary
The N-nitroso compounds (NNC) are recognized as one of the most potent chemical mutagens and carcinogens present in environment and in food [1]. N-nitrosamines are NNC found in foodstuffs, drinking water, rubber products, drug formulations, tobacco and tobacco smoke [2]. Their precursors, nitrites and secondary amines, contained in many common foods, can react under acidic conditions of the stomach and are produced in vivo by reduction of nitrates by bacteria [3]. Toxic effect of chemicals can lead to passive cell death or necrosis, or result in the active mechanism of apoptosis. Activation of the caspase cascade leads to changes in the plasma-membrane, mitochondria and nucleus [17]. Among the family of ten or more different caspases, already described, caspase-8 and -9 are involved in receptor mediated and intracellular (mitochondrial) pathways of apoptotic cascade, respectively.
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