Ethanol-mediated DNA damage and PARP-1 apoptotic responses in cultured fetal cortical neurons.
BACKGROUND
Prior studies by many laboratories have illustrated that ethanol can elicit a cascade of caspase-dependent apoptotic events in cultured neurons. Studies in our laboratory have connected this to oxidative stress and effects on fetal cortical neuron glutathione homeostasis.


OBJECTIVE
The intent of the following studies is to address mechanisms underlying ethanol-associated DNA damage that may be connected to apoptotic death of neurons.


METHODS
Cultures of fetal rat cerebral cortical neurons were utilized. Estimates of DNA damage was determined by Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining and nuclear condensation; Poly(ADP-ribose) polymerase-1 (PARP-1) expression was determined by immunostaining and Western blotting; and occurrence of parylation and AIF translocations were assessed by Western blotting.


RESULTS
Ethanol treatment of the neurons generated increases in DNA damage by 4 hours while nuclear condensation was low at the short exposure period but increased markedly by 24 hours. This was temporally related to a marked up-regulation of PARP-1 expression. Activity of PARP-1, as assessed by PolyADP-ribose (PAR) formation, occurred within 15 minutes and peaked by 6 to 8 hours of ethanol treatment. An almost complete translocation of apoptosis inducing factor (AIF) from mitochondria to the nucleus occurred by 24 hours of ethanol treatment (4.0 mg/ml). Ethanol treatment for 4, 12, and 24 hours elicited an increasing caspase-mediated cleavage of PARP-1 to its 24 kDa fragment.


CONCLUSIONS
These data illustrate the rapid occurrence of DNA damage following ethanol exposure and that PARP-1 pathways may play a role in the subsequent apoptotic death of these neurons.
Cherian
Priscilla P
PP
Department of Medicine, Division of GI/Nutrition, University of Texas Health Science Centre, San Antonio, Texas 78229-3900, USA.
Schenker
Steven
S
Henderson
George I
GI
D000818:Animals; D017209:Apoptosis / Q000187:drug effects / Q000502:physiology; D051033:Apoptosis Inducing Factor / Q000378:metabolism*; D002467:Cell Nucleus / Q000378:metabolism; D002478:Cells, Cultured; D002492:Central Nervous System Depressants / Q000494:pharmacology*; D002540:Cerebral Cortex / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D004249:DNA Damage / Q000187:drug effects*; D000431:Ethanol / Q000494:pharmacology*; D009474:Neurons / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D000071137:Poly (ADP-Ribose) Polymerase-1; D011065:Poly(ADP-ribose) Polymerases / Q000378:metabolism*; D051381:Rats; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology; D013997:Time Factors
D051033:Apoptosis Inducing Factor; D002492:Central Nervous System Depressants; D000431:Ethanol; C499322:Parp1 protein, rat; D000071137:Poly (ADP-Ribose) Polymerase-1; D011065:Poly(ADP-ribose) Polymerases