Carbamylation and oxidation of proteins lead to apoptotic death of lymphocytes

- Carbamylation and oxidation lead to a decrease of mitochondrial potential.
- Both of these processes cause an increase of caspases 2,3 and 9 activities.
- Carbamylation mainly leads to apoptosis, while hydrogen peroxide to necrosis of cells.
- The combination of cyanate and hydrogen peroxide leads to an increase of amounts of necrotic cells.

The apoptotic/necrotic changes in isolated human peripheral blood mononuclear cells (MNCs) subjected to hydrogen peroxide (H2O2), cyanate (NaOCN) and their combination were examined. The mitochondrial potential (ΔΨm), the activities of caspases (−2, −3, −6, −8 and −9) and the level of carbonyls and amino groups in proteins were determined and DNA fragmentation. Apoptotic or necrotic cells were identified by fluorescence microscopy using double staining with Hoechst 33258/propidium iodide.Treatment of MNCs with NaOCN (1 mmol/L and 2 mmol/L), alone and in combination with H2O2 (100 μmol/L), led to a significant decrease in the content of amine groups and a significant increase in the carbonyl level of MNCs in comparison with the control. Measurements taken at three time points (30, 60 and 150 min) showed a significant decrease in ΔΨm in MNCs incubated with H2O2, cyanate and their combination. The highest decrease in ΔΨm was observed after 150 min, when a combination of NaOCN and H2O2 was applied.We observed significant increases in the activities of caspases-2 and -3 in cells exposed to H2O2 and the combination of NaOCN and H2O2. An increase in caspase-2 but not in caspase-3 activity was noted in cells incubated with cyanate. A significant increase in caspase-9 activity in MNCs was observed in all arrangements of tested compounds in comparison with the control.In H2O2-treated cells, a higher level of necrotic cells was noted in comparison to apoptotic cells, whereas carbamylation led mainly to apoptotic cell death. The combination of cyanate and H2O2 increased the population of necrotic cells.