Rat germinal cells require PARP for repair of DNA damage induced by gamma-irradiation and H2O2 treatment.
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.
Atorino
L
L
Department of Organic and Biological Chemistry, University  Federico II of Naples, Italy.
Di Meglio
S
S
Farina
B
B
Jones
R
R
Quesada
P
P
D000818:Animals; D001549:Benzamides / Q000494:pharmacology; D015153:Blotting, Western; D002374:Catalase / Q000378:metabolism; D002467:Cell Nucleus / Q000378:metabolism; D002478:Cells, Cultured; D020552:Comet Assay; D004249:DNA Damage / Q000187:drug effects / Q000235:genetics* / Q000528:radiation effects; D004260:DNA Repair / Q000187:drug effects / Q000235:genetics / Q000528:radiation effects; D004305:Dose-Response Relationship, Drug; D004791:Enzyme Inhibitors; D005720:Gamma Rays / Q000009:adverse effects*; D005854:Germ Cells / Q000187:drug effects* / Q000528:radiation effects*; D005978:Glutathione / Q000031:analogs & derivatives* / Q000494:pharmacology; D006861:Hydrogen Peroxide / Q000009:adverse effects*; D008297:Male; D008856:Microscopy, Fluorescence; D008981:Molsidomine / Q000031:analogs & derivatives / Q000494:pharmacology; D018696:Neuroprotective Agents / Q000494:pharmacology; D020030:Nitric Oxide Donors / Q000494:pharmacology; D009603:Nitroso Compounds / Q000494:pharmacology; D011065:Poly(ADP-ribose) Polymerases / Q000502:physiology*; D051381:Rats; D017208:Rats, Wistar; D026422:S-Nitrosoglutathione; D013087:Spermatids / Q000378:metabolism; D013090:Spermatocytes / Q000378:metabolism; D013482:Superoxide Dismutase / Q000378:metabolism; D013997:Time Factors
D001549:Benzamides; D004791:Enzyme Inhibitors; D018696:Neuroprotective Agents; D020030:Nitric Oxide Donors; D009603:Nitroso Compounds; D026422:S-Nitrosoglutathione; C002385:linsidomine; C025160:3-aminobenzamide; D006861:Hydrogen Peroxide; D008981:Molsidomine; D002374:Catalase; D013482:Superoxide Dismutase; D011065:Poly(ADP-ribose) Polymerases; D005978:Glutathione