title: Senescence of primary amniotic cells via oxidative DNA damage.
abstract: OBJECTIVE
  Oxidative stress is a postulated etiology of spontaneous preterm birth (PTB) and preterm prelabor rupture of the membranes (pPROM); however, the precise mechanistic role of reactive oxygen species (ROS) in these complications is unclear. The objective of this study is to examine impact of a water soluble cigarette smoke extract (wsCSE), a predicted cause of pregnancy complications, on human amnion epithelial cells.
  
  
  METHODS
  Amnion cells isolated from fetal membranes were exposed to wsCSE prepared in cell culture medium and changes in ROS levels, DNA base and strand damage was determined by using 2'7'-dichlorodihydro-fluorescein and comet assays as well as Fragment Length Analysis using Repair Enzymes (FLARE) assays, respectively. Western blot analyses were used to determine the changes in mass and post-translational modification of apoptosis signal-regulating kinase (ASK1), phospho-p38 (P-p38 MAPK), and p19(arf). Expression of senescence-associated β-galectosidase (SAβ-gal) was used to confirm cell ageing in situ.
  
  
  RESULTS
  ROS levels in wsCSE-exposed amnion cells increased rapidly (within 2 min) and significantly (p<0.01) at all-time points, and DNA strand and base damage was evidenced by comet and FLARE assays. Activation of ASK1, P-p38 MAPK and p19(Arf) correlated with percentage of SAβ-gal expressing cells after wsCSE treatment. The antioxidant N-acetyl-L-cysteine (NAC) prevented ROS-induced DNA damage and phosphorylation of p38 MAPK, whereas activation of ASK1 and increased expression of p19(Arf) were not significantly affected by NAC.
  
  
  CONCLUSIONS
  The findings support the hypothesis that compounds in wsCSE induces amnion cell senescence via a mechanism involving ROS and DNA damage. Both pathways may contribute to PTB and pPROM. Our results imply that antioxidant interventions that control ROS may interrupt pathways leading to pPROM and other causes of PTB.
authors.affiliation: Department of Obstetrics and Gynecology, Division of Maternal-Fetal Medicine Perinatal Research, The University of Texas Medical Branch at Galveston, Galveston, Texas, United States of America.
authors.forename: Ramkumar
authors.initials: R
authors.lastname: Menon
authors.affiliation: Department of Microbiology and Immunology, The University of Texas Medical Branch at Galveston, Galveston, Texas, United States of America.
authors.forename: Istvan
authors.initials: I
authors.lastname: Boldogh
authors.affiliation: Department of Obstetrics and Gynecology, Division of Maternal-Fetal Medicine Perinatal Research, The University of Texas Medical Branch at Galveston, Galveston, Texas, United States of America.
authors.forename: Rheanna
authors.initials: R
authors.lastname: Urrabaz-Garza
authors.affiliation: Department of Obstetrics and Gynecology, Division of Maternal-Fetal Medicine Perinatal Research, The University of Texas Medical Branch at Galveston, Galveston, Texas, United States of America.
authors.forename: Jossimara
authors.initials: J
authors.lastname: Polettini
authors.affiliation: Department of Obstetrics and Gynecology, Division of Maternal-Fetal Medicine Perinatal Research, The University of Texas Medical Branch at Galveston, Galveston, Texas, United States of America.
authors.forename: Tariq Ali
authors.initials: TA
authors.lastname: Syed
authors.affiliation: Department of Obstetrics and Gynecology, Division of Maternal-Fetal Medicine Perinatal Research, The University of Texas Medical Branch at Galveston, Galveston, Texas, United States of America.
authors.forename: George R
authors.initials: GR
authors.lastname: Saade
authors.affiliation: Department of Biochemistry and Molecular Biology, The University of Texas Medical Branch at Galveston, Galveston, Texas, United States of America.
authors.forename: John
authors.initials: J
authors.lastname: Papaconstantinou
authors.affiliation: Department of Obstetrics and Gynecology, Wake Forest School of Medicine, Winston-Salem, North Carolina, United States of America.
authors.forename: Robert N
authors.initials: RN
authors.lastname: Taylor
mesh_terms: D000650:Amnion / Q000378:metabolism*; D016922:Cellular Senescence / Q000235:genetics*; D004249:DNA Damage; D004847:Epithelial Cells / Q000187:drug effects / Q000378:metabolism*; D005260:Female; D006801:Humans; D048848:MAP Kinase Kinase Kinase 5 / Q000378:metabolism; D018384:Oxidative Stress; D010766:Phosphorylation; D011247:Pregnancy; D017382:Reactive Oxygen Species / Q000378:metabolism; D014026:Nicotiana / Q000737:chemistry; D001616:beta-Galactosidase / Q000378:metabolism; D048051:p38 Mitogen-Activated Protein Kinases
chemical_list: D017382:Reactive Oxygen Species; D048051:p38 Mitogen-Activated Protein Kinases; D048848:MAP Kinase Kinase Kinase 5; C482858:MAP3K5 protein, human; D001616:beta-Galactosidase