Senescence of primary amniotic cells via oxidative DNA damage

Ramkumar Menon, Istvan Boldogh, Rheanna Urrabaz-Garza, Jossimara Polettini, Tariq Ali Syed, George Saade, John Papaconstantinou, Robert N. Taylor

Research output: Contribution to journalArticle

43 Citations (Scopus)

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 p19arf. 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 p19Arf 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.

Original languageEnglish (US)
Article numbere83416
JournalPLoS One
Volume8
Issue number12
DOIs
StatePublished - Dec 27 2013

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cigarettes
DNA damage
amnion
DNA Damage
smoke
reactive oxygen species
Reactive Oxygen Species
Amnion
Smoke
Tobacco Products
premature birth
DNA
Premature Birth
p38 Mitogen-Activated Protein Kinases
mitogen-activated protein kinase
Water
extracts
Membranes
Rupture
Assays

ASJC Scopus subject areas

  • Agricultural and Biological Sciences(all)
  • Biochemistry, Genetics and Molecular Biology(all)
  • Medicine(all)

Cite this

Senescence of primary amniotic cells via oxidative DNA damage. / Menon, Ramkumar; Boldogh, Istvan; Urrabaz-Garza, Rheanna; Polettini, Jossimara; Syed, Tariq Ali; Saade, George; Papaconstantinou, John; Taylor, Robert N.

In: PLoS One, Vol. 8, No. 12, e83416, 27.12.2013.

Research output: Contribution to journalArticle

Menon, Ramkumar ; Boldogh, Istvan ; Urrabaz-Garza, Rheanna ; Polettini, Jossimara ; Syed, Tariq Ali ; Saade, George ; Papaconstantinou, John ; Taylor, Robert N. / Senescence of primary amniotic cells via oxidative DNA damage. In: PLoS One. 2013 ; Vol. 8, No. 12.
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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 p19arf. 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 p19Arf 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.",
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