Mitochondrial dysfunction and death in motor neurons exposed to the glutathione-depleting agent ethacrynic acid.
This study investigated the mechanisms of toxicity of glutathione (GSH) depletion in one cell type, the motor neuron. Ethacrynic acid (EA) (100 microM) was added to immortalized mouse motor neurons (NSC-34) to deplete both cytosolic and mitochondrial glutathione rapidly. This caused a drop in GSH to 25% of the initial level in 1 h and complete loss in 4 h. This effect was accompanied by enhanced generation of reactive oxygen species (ROS) with a peak after 2 h of exposure, and by signs of mitochondrial dysfunction such as a decrease in 3-(4,5-dimethyl-2-thiazoyl)-2,5-diphenyltetrazolium bromide (MTT) (30% less after 4 h). The increase in ROS and the MTT reduction were both EA concentration-dependent. Expression of heme oxygenase-1 (HO-1), a marker of oxidative stress, also increased. The mitochondrial damage was monitored by measuring the mitochondrial membrane potential (MMP) from the uptake of rhodamine 123 into mitochondria. MMP dropped (20%) after only 1 h exposure to EA, and slowly continued to decline until 3 h, with a steep drop at 5 h (50% decrease), i.e. after the complete GSH loss. Quantification of DNA fragmentation by the TUNEL technique showed that the proportion of cells with fragmented nuclei rose from 10% after 5 h EA exposure to about 65% at 18 h. These results indicate that EA-induced GSH depletion rapidly impairs the mitochondrial function of motor neurons, and this precedes cell death. This experimental model of oxidative toxicity could be useful to study mechanisms of diseases like spinal cord injury (SCI) and amyotrophic lateral sclerosis (ALS), where motor neurons are the vulnerable population and oxidative stress has a pathogenic role.
Rizzardini
M
M
Istituto di Ricerche Farmacologiche Mario Negri, Via Eritrea 62, 20157 Milan, Italy.
Lupi
M
M
Bernasconi
S
S
Mangolini
A
A
Cantoni
L
L
D000818:Animals; D002460:Cell Line; D004305:Dose-Response Relationship, Drug; D004976:Ethacrynic Acid / Q000633:toxicity*; D005978:Glutathione / Q000378:metabolism*; D008564:Membrane Potentials / Q000187:drug effects / Q000502:physiology; D051379:Mice; D008928:Mitochondria / Q000187:drug effects* / Q000378:metabolism; D028361:Mitochondrial Diseases / Q000139:chemically induced / Q000378:metabolism; D009046:Motor Neurons / Q000187:drug effects* / Q000378:metabolism; D014407:Tumor Cells, Cultured
D005978:Glutathione; D004976:Ethacrynic Acid