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Delayed induction of apoptosis by ammonia in C6 glioma cells
L Buzańska1, B Zabłocka, A Dybel
1Department of Neurotoxicology, Medical Research Centre, Polish Academy of Sciences, Warsaw. buzanska@cmdik.pan.pl
Abstract:
Ammonia is a neurotoxin whose administration in large doses causes coma and death of the exposed animals, but whether and in what degree these whole body effects are related to the death of CNS cells is not known. Since the downstream effects of ammonia in cultured CNS cells appear to be partly mediated by overactivation of several putative signalling mechanisms characteristic for the apoptotic program, we speculated that ammonia neurotoxicity may be apoptogenic. In this study, C6 glioma cells grown in 2% serum were exposed to 5 mM or 10 mM NH(4)Cl (ammonia) for 96 h and tested for the appearance of apoptosis by (a) Hoechst staining, (b) TUNEL reaction and (c) DNA ladder, at different times of exposure. In cultures exposed to either 5 mM or 10 mM ammonia, about 10% of the cells were found to enter apoptosis at 48 h of exposure, and the number of apoptotic cells rose to 30% at 72 h, and to 50% at 96 h of exposure, respectively. The first transduction signal purportedly involved in apoptosis, activation of PKCalphabeta, was transient and appeared already after 3-6 h of treatment. Coincident with pronounced manifestation of apoptosis (at 72 h and even more at 96 h of exposure) was an increased transfer of the transcription factor NFkappaB from cytoplasmto nucleus as revealed by EMSA assay. The number of cells affected by ammonia-induced apoptosis was markedly reduced by incubation with a NOS inhibitor, L-NAME at 100 microM concentration. The results indicate that ammonia-induced apoptosis is a result of a complex interplay of at least three signalling molecules: NO, PKC and the transcription factor NFkappaB, with NFkappaB being possibly involved in the induction of iNOS and generation of toxic levels of NO in the cells.
Insights
Ammonia exposure induces apoptosis in CNS cells, a process involving nitric oxide (NO), protein kinase C (PKC), and nuclear factor kappa B (NFkappaB). This research clarifies ammonia
Area of Science:
- Neuroscience
- Cell Biology
- Toxicology
Background:
- Ammonia is a known neurotoxin, but its precise role in central nervous system (CNS) cell death is unclear.
- Ammonia's effects on cultured CNS cells suggest involvement of apoptotic signaling pathways.
Purpose of the Study:
- To investigate whether ammonia exposure induces apoptosis in C6 glioma cells.
- To identify the signaling molecules involved in ammonia-induced apoptosis.
Main Methods:
- C6 glioma cells were exposed to 5 mM or 10 mM ammonia (NH4Cl) for 96 hours.
- Apoptosis was assessed using Hoechst staining, TUNEL reaction, and DNA ladder assays.
- Signaling pathway activation was analyzed via PKC assays, EMSA for NFkappaB, and effects of NOS inhibitor L-NAME.
Main Results:
- Ammonia exposure led to a dose- and time-dependent increase in apoptosis, reaching 50% by 96 hours.
- Early activation of PKC and later translocation of NFkappaB to the nucleus were observed.
- The NOS inhibitor L-NAME significantly reduced ammonia-induced apoptosis.
Conclusions:
- Ammonia induces apoptosis in CNS cells through a complex signaling cascade.
- Nitric oxide (NO), PKC, and NFkappaB are key mediators of ammonia neurotoxicity.
- NFkappaB may induce iNOS, leading to toxic NO levels and subsequent cell death.