title: The activity and inhibition of poly(ADP-ribose) polymerase-1 in equine peripheral blood mononuclear cells in vitro.
abstract: OBJECTIVE
  To evaluate the poly (ADP-ribose) polymerase-1 (PARP1) enzyme and its inhibition in horses and explore its potential as a novel therapeutic target for equine intestinal ischemia-reperfusion injury by (1) identifying poly (ADP-ribose) (PAR) as an indication of PARP1 activation in equine cells using available immunoblot analytical techniques, (2) inducing PARP1 activation in an in vitro oxidative DNA damage model, (3) and demonstrating the inhibition of PARP1 in equine cells using commercially available PARP1 inhibitors.
  
  
  METHODS
  Experimental study.
  
  
  METHODS
  Blood samples were collected from systemically healthy ponies (n = 3) and horses (n = 3).
  
  
  METHODS
  (1) Equine peripheral blood mononuclear cells were exposed to 3 different concentrations of hydrogen peroxide (H2 O2 ) and were lysed at specific time points. PARP1 activity was then assessed by using immunoblot analyses to determine PAR levels. (2) Equine peripheral blood mononuclear cells were preincubated with defined concentrations of PARP1 inhibitors prior to H2 O2 -mediated PARP1 stimulation. PAR levels reflecting PARP1 activity were determined using immunoblot analyses.
  
  
  RESULTS
  Commercially available anti-PAR antibodies were used successfully to identify equine PAR. There was a significant increase in PAR accumulation following treatment with H2 O2 . All of the tested PARP inhibitors significantly reduced PAR accumulation to or below basal levels following treatment with H2 O2 .
  
  
  CONCLUSIONS
  This proof of principle study demonstrated that PAR, an indicator of PARP1 activity, can be identified in the equine species using immunoblot techniques, that equine PARP1 can be activated by H2 O2 -induced DNA damage, and that this activation can be inhibited by PARP1 enzyme inhibitors. The data suggest that the PARP1 pathway plays a role in the equine cellular response to oxidative DNA damage and supports its potential as a novel therapeutic target. Further research documenting an increase in PAR levels in vivo and the efficacy of PARP1 inhibitors in an equine intestinal ischemia-reperfusion model is needed.
authors.affiliation: School of Veterinary Medicine, University of Pennsylvania, Philadelphia, PA, 19104.
authors.forename: Hope F
authors.initials: HF
authors.lastname: Douglas
authors.affiliation: Department of Clinical Studies, New Bolton Center, University of Pennsylvania, Kennett Square, PA, 19348.
authors.forename: Louise L
authors.initials: LL
authors.lastname: Southwood
authors.affiliation: Department of Animal Biology, University of Pennsylvania, Philadelphia, PA, 19104.
authors.forename: Mirella L
authors.initials: ML
authors.lastname: Meyer-Ficca
authors.affiliation: Department of Clinical Studies, New Bolton Center, University of Pennsylvania, Kennett Square, PA, 19348.
authors.forename: Samantha K
authors.initials: SK
authors.lastname: Hart
authors.affiliation: Department of Animal Biology, University of Pennsylvania, Philadelphia, PA, 19104.
authors.forename: Ralph G
authors.initials: RG
authors.lastname: Meyer
mesh_terms: D000818:Animals; D006734:Horse Diseases / Q000503:physiopathology; D006736:Horses / Q000097:blood*; D066298:In Vitro Techniques; D045822:Intestinal Volvulus / Q000503:physiopathology / Q000662:veterinary; D007963:Leukocytes, Mononuclear / Q000187:drug effects* / Q000201:enzymology; D000067856:Poly(ADP-ribose) Polymerase Inhibitors / Q000494:pharmacology*; D011065:Poly(ADP-ribose) Polymerases / Q000378:metabolism*; D015427:Reperfusion Injury / Q000503:physiopathology / Q000662:veterinary
chemical_list: D000067856:Poly(ADP-ribose) Polymerase Inhibitors; D011065:Poly(ADP-ribose) Polymerases
keywords: biomarker; horse; ischemia-reperfusion injury; oxidative stress