Early acute necrosis, delayed apoptosis and cytoskeletal breakdown in cultured cerebellar granule neurons exposed to methylmercury.
Cerebellar granule cells (CGCs) are a sensitive target for methylmercury (MeHg) neurotoxicity. In vitro exposure of primary cultures of rat CGCs to MeHg resulted in a time- and concentration-dependent cell death. Within 1 hr exposure, MeHg at 5-10 microM caused impairment of mitochondrial activity, de-energization of mitochondria and plasma membrane lysis, resulting in necrotic cell death. Lower MeHg concentrations (0.5-1 microM) did not compromise cell viability, mitochondrial membrane potential and function at early time points. Later, however, the cells progressively underwent apoptosis and 100% cell death was reached by 18 hr treatment. Neuronal network fragmentation and microtubule depolymerization were detected as early as within 1.5 hr of MeHg (1 microM) exposure, long before the occurrence of nuclear condensation (6-9 hr). Neurite damage worsened with longer exposure time and proceeded to the complete dissolution of microtubules and neuronal processes (18 hr). Microtubule stabilization by taxol did not prevent MeHg-induced delayed apoptosis. Similarly ineffective were the caspase inhibitors z-VAD-fluoromethylketone and z-DEVD-chloromethylketone, the L-type calcium channel inhibitor nifedipine, the calcium chelator EGTA and BAPTA, and the NMDA receptor antagonist MK-801. On the other hand, insulin-like growth factor-I partially rescued CGCs from MeHg-triggered apoptosis. Altogether these results provide evidence that the intensity of MeHg insult is decisive in the time of onset and the mode of neuronal death that follows, i.e., necrosis vs. apoptosis, and suggest that cytoskeletal breakdown and deprivation of neurotrophic support play a role in MeHg delayed toxicity.
Castoldi
A F
AF
Toxicology Research Centre, Salvatore Maugeri Foundation, IRCCS, San Martino Siccomario, Pavia, Italy. acastoldi@fsm.it
Barni
S
S
Turin
I
I
Gandini
C
C
Manzo
L
L
D000818:Animals; D000831:Animals, Newborn; D018929:Cell Culture Techniques; D016923:Cell Death / Q000187:drug effects*; D002531:Cerebellum / Q000187:drug effects* / Q000648:ultrastructure; D003599:Cytoskeleton / Q000187:drug effects*; D008767:Methylmercury Compounds / Q000633:toxicity*; D008854:Microscopy, Electron; D008870:Microtubules / Q000187:drug effects; D009474:Neurons / Q000187:drug effects* / Q000648:ultrastructure*; D051381:Rats; D017207:Rats, Sprague-Dawley; D013997:Time Factors
D008767:Methylmercury Compounds