Rat germinal cells require PARP for repair of DNA damage induced by gamma-irradiation and H2O2 treatment

Eur J Cell Biol. 2001 Mar;80(3):222-9. doi: 10.1078/0171-9335-00153.

Abstract

The ability of rat germinal cells to recover from genotoxic stress has been investigated using isolated populations of primary spermatocytes and round spermatids. Using a comet assay at pH 10.0 to assess single strand breakage (SSB) in DNA, it was found that a high level of damage was induced by 5 Gy gamma-irradiation and acute exposure to 50 microM H2O2. This damage was effectively repaired during a subsequent recovery period of 1-3 hours culture in vitro but repair was significantly delayed in the presence of the poly(ADP-ribose)polymerase (PARP) inhibitor 3-aminobenzamide (3-ABA). Immunofluorescence detection of PARP with specific antibodies localised the protein to discrete foci within the nucleus of both spermatocytes and spermatids. Poly(ADP-ribose) (pADPR) could also be detected in spermatid nuclei following gamma-irradiation or H2O2 treatment. Moreover, PARP activation occurs both in spermatocytes and spermatids left to recover after both genotoxic stresses. The NO donors, 3-morpholino-sydnonimine (SIN-1) and S-nitrosoglutathione (SNOG), caused significant SSBs in both spermatocytes and spermatids. The effects of SIN-1 could be prevented by exogenous catalase (CAT), but not superoxide dismutase (SOD), in the cell suspensions. SNOG-induced SSBs were insensitive to both CAT and SOD. It is concluded that DNA in spermatocytes and spermatids is sensitive to damage by gamma-irradiation and H2O2 and that efficient repair of SSBs requires PARP activity.

MeSH terms

  • Animals
  • Benzamides / pharmacology
  • Blotting, Western
  • Catalase / metabolism
  • Cell Nucleus / metabolism
  • Cells, Cultured
  • Comet Assay
  • DNA Damage / drug effects
  • DNA Damage / genetics*
  • DNA Damage / radiation effects
  • DNA Repair / drug effects
  • DNA Repair / genetics
  • DNA Repair / radiation effects
  • Dose-Response Relationship, Drug
  • Enzyme Inhibitors
  • Gamma Rays / adverse effects*
  • Germ Cells / drug effects*
  • Germ Cells / radiation effects*
  • Glutathione / analogs & derivatives*
  • Glutathione / pharmacology
  • Hydrogen Peroxide / adverse effects*
  • Male
  • Microscopy, Fluorescence
  • Molsidomine / analogs & derivatives
  • Molsidomine / pharmacology
  • Neuroprotective Agents / pharmacology
  • Nitric Oxide Donors / pharmacology
  • Nitroso Compounds / pharmacology
  • Poly(ADP-ribose) Polymerases / physiology*
  • Rats
  • Rats, Wistar
  • S-Nitrosoglutathione
  • Spermatids / metabolism
  • Spermatocytes / metabolism
  • Superoxide Dismutase / metabolism
  • Time Factors

Substances

  • Benzamides
  • Enzyme Inhibitors
  • Neuroprotective Agents
  • Nitric Oxide Donors
  • Nitroso Compounds
  • S-Nitrosoglutathione
  • linsidomine
  • 3-aminobenzamide
  • Hydrogen Peroxide
  • Molsidomine
  • Catalase
  • Superoxide Dismutase
  • Poly(ADP-ribose) Polymerases
  • Glutathione