title: Phospholipase A2, oxidative stress, and neurodegeneration in binge ethanol-treated organotypic slice cultures of developing rat brain.
abstract: BACKGROUND
  Brain neurodamage from chronic binge ethanol (EtOH) exposure is linked to neuroinflammation and associated oxidative stress. Using rat organotypic hippocampal-entorhinal cortical (HEC) slice cultures of developing brain age, we reported that binge EtOH promotes release of a neuroinflammatory instigator, arachidonic acid (AA), concomitant with neurodegeneration, and that mepacrine, a global inhibitor of phospholipase A2 (PLA2) enzymes mobilizing AA from phospholipids, is neuroprotective. Here, we sought with binge EtOH-treated HEC cultures to establish that PLA2 activity is responsible in part for significant oxidative stress and to ascertain the PLA2 families responsible for AA release and neurodegeneration.
  
  
  METHODS
  HEC slices, prepared from 1-week-old rats and cultured 2 to 2.5 weeks, were exposed to 100 mM EtOH over 6 successive days, with 4 daytime "withdrawals" (no EtOH). Brain 3-nitrotyrosinated (3-NT)- and 4-hydroxy-2-nonenal (4-HNE)-adducted proteins, oxidative stress footprints, were immunoassayed on days 3 through 6, and mepacrine's effect was determined on day 6. The effects of specific PLA2 inhibitors on neurodegeneration (propidium iodide staining) and AA release (ELISA levels in media) in the cultures were then determined. Also, the effect of JZL184, an inhibitor of monoacylglycerol lipase (MAGL) which is reported to mobilize AA from endocannabinoids during neuroinflammatory insults, was examined.
  
  
  RESULTS
  3-NT- and 4-HNE-adducted proteins were significantly increased by the binge EtOH exposure, consistent with oxidative stress, and mepacrine prevented the increases. The PLA2 inhibitor results implicated secretory PLA2 (group II sPLA2) and to some extent Ca(2+) -independent cytosolic PLA2 (group VI iPLA2) in binge EtOH-induced neurotoxicity and in AA release, but surprisingly, Ca(2+) -dependent cytosolic PLA2 (group IV cPLA2) did not appear important. Furthermore, unlike PLA2 inhibition, MAGL inhibition failed to prevent the neurodegeneration.
  
  
  CONCLUSIONS
  In these developing HEC slice cultures, pro-oxidative signaling via sPLA2 and iPLA2, but not necessarily cPLA2 or MAGL, is involved in EtOH neurotoxicity. This study provides further insights into neuroinflammatory phospholipase signaling and oxidative stress underlying binge EtOH-induced neurodegeneration in developing (adolescent age) brain in vitro.
authors.affiliation: Department of Molecular Pharmacology & Therapeutics , Stritch School of Medicine, Loyola University Chicago, Maywood, Illinois.
authors.forename: Kwan-Hoon
authors.initials: KH
authors.lastname: Moon
authors.forename: Nuzhath
authors.initials: N
authors.lastname: Tajuddin
authors.forename: James
authors.initials: J
authors.lastname: Brown
authors.forename: Edward J
authors.initials: EJ
authors.lastname: Neafsey
authors.forename: Hee-Yong
authors.initials: HY
authors.lastname: Kim
authors.forename: Michael A
authors.initials: MA
authors.lastname: Collins
mesh_terms: D000818:Animals; D000831:Animals, Newborn; D063425:Binge Drinking / Q000378:metabolism*; D018728:Entorhinal Cortex / Q000187:drug effects / Q000254:growth & development / Q000378:metabolism*; D004789:Enzyme Activation / Q000187:drug effects / Q000502:physiology; D000431:Ethanol / Q000633:toxicity*; D006624:Hippocampus / Q000187:drug effects / Q000254:growth & development / Q000378:metabolism*; D019636:Neurodegenerative Diseases / Q000139:chemically induced / Q000378:metabolism; D009924:Organ Culture Techniques; D018384:Oxidative Stress / Q000187:drug effects / Q000502:physiology*; D054467:Phospholipases A2 / Q000096:biosynthesis*; D051381:Rats; D017207:Rats, Sprague-Dawley
chemical_list: D000431:Ethanol; D054467:Phospholipases A2
keywords: Alcohol; Arachidonic Acid; Endocannabinoid; Monoacylglycerol Lipase; Neuroinflammation; Organotypic
references.citation: Neurosci Lett. 2003 Feb 27;338(2):164-8
references.pmid: 12566178
references.citation: Int J Mol Med. 2008 Jun;21(6):737-45
references.pmid: 18506367
references.citation: Alcohol Clin Exp Res. 2006 Nov;30(11):1938-49
references.pmid: 17067360
references.citation: Alcohol. 1993 Nov-Dec;10(6):559-61
references.pmid: 8123218
references.citation: Biochim Biophys Acta. 2000 Oct 31;1488(1-2):28-39
references.pmid: 11080674
references.citation: Neurochem Res. 2009 Feb;34(2):260-7
references.pmid: 18592376
references.citation: J Neuroimmune Pharmacol. 2010 Dec;5(4):533-45
references.pmid: 20379791
references.citation: Alcohol Clin Exp Res. 1996 Apr;20(2):284-92
references.pmid: 8730219
references.citation: Biochem Biophys Res Commun. 1992 May 29;185(1):350-5
references.pmid: 1599471
references.citation: J Biol Chem. 1998 May 29;273(22):13870-7
references.pmid: 9593733
references.citation: Int J Cancer. 2008 Apr 15;122(8):1757-64
references.pmid: 18076063
references.citation: J Neurosci. 2010 Jun 16;30(24):8285-95
references.pmid: 20554880
references.citation: J Pharmacol Exp Ther. 2005 Aug;314(2):780-8
references.pmid: 15878999
references.citation: Biochemistry. 1993 Jun 15;32(23):5935-40
references.pmid: 8018213
references.citation: J Stud Alcohol. 1991 Sep;52(5):448-53
references.pmid: 1943100
references.citation: J Neuroinflammation. 2008 Mar 18;5:10
references.pmid: 18348728
references.citation: Front Biosci (Elite Ed). 2012 Jan 01;4:1358-67
references.pmid: 22201960
references.citation: J Biol Chem. 1995 Jan 6;270(1):445-50
references.pmid: 7814408
references.citation: Science. 1995 Oct 6;270(5233):99-102
references.pmid: 7569957
references.citation: Brain Behav Immun. 2011 Jun;25 Suppl 1:S129-36
references.pmid: 21262340
references.citation: Alcohol Clin Exp Res. 2002 Apr;26(4):558-64
references.pmid: 11981133
references.citation: Br J Psychiatry. 2008 Nov;193(5):351-3
references.pmid: 18978310
references.citation: Hepatology. 2006 Nov;44(5):1218-30
references.pmid: 17058263
references.citation: Science. 2011 Nov 11;334(6057):809-13
references.pmid: 22021672
references.citation: Curr Drug Targets. 2008 Aug;9(8):683-97
references.pmid: 18691015
references.citation: J Neurochem. 2005 Aug;94(4):980-94
references.pmid: 16092941
references.citation: Biochem Pharmacol. 1998 Feb 15;55(4):515-21
references.pmid: 9514087
references.citation: Free Radic Biol Med. 2006 Feb 1;40(3):364-75
references.pmid: 16443151
references.citation: J Neurochem. 2008 Mar;104(5):1177-89
references.pmid: 17986230
references.citation: J Biol Chem. 2001 Apr 13;276(15):12035-40
references.pmid: 11134037
references.citation: Chem Biol. 2012 May 25;19(5):579-88
references.pmid: 22542104
references.citation: Neurochem Int. 2008 Feb;52(3):487-94
references.pmid: 17900761
references.citation: Biom J. 2008 Jun;50(3):346-63
references.pmid: 18481363
references.citation: Immunopharmacology. 1993 Jul-Aug;26(1):1-9
references.pmid: 8407280
references.citation: Biochim Biophys Acta. 2003 Nov 30;1635(1):37-47
references.pmid: 14642775
references.citation: J Toxicol Clin Toxicol. 1989;27(6):375-84
references.pmid: 2628586
references.citation: Acta Physiol (Oxf). 2006 May-Jun;187(1-2):75-85
references.pmid: 16734744
references.citation: J Neuroinflammation. 2012 Jan 12;9:5
references.pmid: 22240163
references.citation: J Neurotrauma. 2009 Feb 11;26(2):261-73
references.pmid: 19236167
references.citation: Ann Emerg Med. 2001 Dec;38(6):653-9
references.pmid: 11719745
references.citation: Neuroscience. 2004;124(4):869-77
references.pmid: 15026127
references.citation: J Neuropathol Exp Neurol. 2008 May;67(5):417-27
references.pmid: 18431255
references.citation: J Immunol. 2006 Nov 1;177(9):6398-404
references.pmid: 17056571
references.citation: Biochim Biophys Acta. 1996 Jul 12;1302(1):55-60
references.pmid: 8695655
references.citation: Toxicology. 2009 Aug 21;262(3):199-206
references.pmid: 19540902
references.citation: J Neurophysiol. 1998 Mar;79(3):1441-9
references.pmid: 9497423
references.citation: FASEB J. 1998 Feb;12(2):221-30
references.pmid: 9472987
references.citation: J Neurochem. 1982 Jan;38(1):84-93
references.pmid: 7108537
references.citation: J Neuroinflammation. 2012 Jun 18;9:130
references.pmid: 22709825
references.citation: J Biol Chem. 1985 Jun 25;260(12):7234-40
references.pmid: 3997864
references.citation: Mol Neurobiol. 2012 Aug;46(1):85-95
references.pmid: 22476944
references.citation: Alcohol. 2013 Feb;47(1):39-45
references.pmid: 23102656
references.citation: Exp Neurol. 2008 Sep;213(1):10-7
references.pmid: 18625499
references.citation: Methods Mol Biol. 2007;399:55-66
references.pmid: 18309925
references.citation: Brain Res. 2003 Oct 24;988(1-2):20-8
references.pmid: 14519523