benzyloxycarbonylvalyl-alanyl-aspartyl-fluoromethyl-ketone and trimethyltin

Trimethyltin (TMT) produces selective neuronal degeneration in the central nervous system (CNS), in which the hippocampus is the most sensitive area. Since previous studies have been conducted in either non-neural cells or mixed primary cultures, an immortalized hippocampal neuronal cell line (HT-22 cell) was used to assess the mechanism and mode of death produced by TMT. The compound produced a time- and concentration-dependent apoptotic death that was caspase-mediated. Excessive generation of reactive oxygen species (ROS) and subsequent reduction of mitochondrial membrane potential (DeltaPsim) were involved in the cytotoxicity. Scavenging of ROS by a free radical trapping agent or inhibition of the mitochondrial permeability transition (MPT) pore significantly reduced cell death. Additionally, TMT increased expression of inducible nitric oxide synthase (iNOS) by activation of the redox-sensitive transcription factor NFkappaB. Pharmacologic inhibition studies showed that the iNOS-mediated NO generation increased expression of Bax and then mitochondrial-mediated apoptosis. It was concluded that excessive ROS generation initiated the apoptotic cell death by upregulating iNOS followed by increased Bax expression which then led to loss of DeltaPsim and caspase-executed cell death. This study is the first to report in a neuronal cell model that TMT stimulates induction of iNOS, which then increases cellular levels of reactive nitrogen species (RNS) to initiate apoptotic death.

Topics: Amino Acid Chloromethyl Ketones; Animals; Apoptosis; bcl-2-Associated X Protein; Caspase 3; Caspase Inhibitors; Cell Line; Cyclic N-Oxides; Cyclosporine; Dose-Response Relationship, Drug; Enzyme Inhibitors; Hippocampus; Membrane Potentials; Mitochondrial Membrane Transport Proteins; Mitochondrial Membranes; Mitochondrial Permeability Transition Pore; NG-Nitroarginine Methyl Ester; Nitric Oxide; Nitric Oxide Synthase Type II; Nitrogen Oxides; Oligonucleotides, Antisense; Peptides; Reactive Oxygen Species; Trimethyltin Compounds; Up-Regulation

Acute exposure to trimethyltin (TMT) causes neuronal degeneration in the hippocampus, amygdala, pyriform cortex, and neocortex [Am. J. Pathol. 97 (1979) 59]. Despite extensive efforts elucidating neuropathological changes and behavioral deficits following TMT exposure, only a limited amount of work has examined the molecular signaling mechanisms that lead to these changes. The present paper demonstrates that TMT impairs neurite outgrowth and cell viability in an in vitro model of neuronal development. The decrease in cell viability is paralleled by a decrease in cell body size, an increase in DNA fragmentation, activation of caspase-9, and cleavage of the caspase substrate poly-ADP ribose polymerase (PARP). These results suggest that TMT induces apoptosis. Pharmacological inhibition of caspase activity, p38 stress-responsive protein kinase activity, or oxidative stress prevented TMT-induced cell death. This work provides the first evidence for a TMT-initiated apoptotic pathway requiring oxidative stress, caspase activation, and p38 protein kinase activity.

Topics: Amino Acid Chloromethyl Ketones; Animals; Apoptosis; Blotting, Western; Caspase 9; Caspase Inhibitors; Caspases; Cell Size; Cell Survival; Cysteine Proteinase Inhibitors; DNA Fragmentation; Dose-Response Relationship, Drug; Mitogen-Activated Protein Kinases; Neurites; Oxidative Stress; p38 Mitogen-Activated Protein Kinases; PC12 Cells; Poly(ADP-ribose) Polymerases; Rats; Trimethyltin Compounds