Protein tyrosine nitration and poly(ADP-ribose) polymerase activation in N-methyl-N-nitro-N-nitrosoguanidine-treated thymocytes: implication for cytotoxicity.
1-Methyl-3-nitro-1-nitrosoguanidine (MNNG) is a DNA alkylating agent. DNA alkylation by MNNG is known to trigger accelerated poly(ADP-ribose) metabolism. Various nitroso compounds release nitric oxide (NO). Therefore, we set out to investigate whether MNNG functions as NO donor and whether MNNG-derived NO or secondary NO metabolites such as peroxynitrite contribute to MNNG-induced cytotoxicity. MNNG in aqueous solutions resulted in time- and concentration-dependent NO release and nitrite/nitrate formation. Moreover, various proteins in MNNG-treated thymocytes were found to be nitrated, indicating that MNNG-derived NO may combine with cellular superoxide to form peroxynitrite, a nitrating agent. MNNG also caused DNA breakage and increased poly(ADP-ribose) polymerase activity and cytotoxicity in thymocytes. MNNG-induced DNA damage (measured by the comet assay) and thymocyte death (measured by propidium iodide uptake) was prevented by the PARP inhibitor PJ-34 and by glutathione (GSH) or N-acetylcysteine (NAC). The cytoprotection provided by PJ-34 against necrotic parameters was paralleled by increased outputs in apoptotic parameters (caspase activity, DNA laddering) indicating that PARP activation diverts apoptotic death toward necrosis. As MNNG-induced cytotoxicity showed many similarities to peroxynitrite-induced cell death, we tested whether peroxynitrite was responsible for at least part of the cytotoxicity induced by MNNG. Cell-permeable enzymic antioxidants (superoxide dismutase and catalase), the NO scavenger cPTIO or the peroxynitrite decomposition catalyst FP15 failed to inhibit MNNG-induced DNA breakage and cytotoxicity. In conclusion, MNNG induces tyrosine nitration in thymocytes. Furthermore, MNNG damages DNA by a radical mechanism that does not involve NO or peroxynitrite.
Bai
Péter
P
Department of Medical Chemistry, MHSC, RCMM, University of Debrecen, H-4032 Debrecen, Nagyerdei krt. 98, Hungary.
Hegedus
Csaba
C
Erdélyi
Katalin
K
Szabó
Eva
E
Bakondi
Edina
E
Gergely
Szabolcs
S
Szabó
Csaba
C
Virág
László
L
D000477:Alkylating Agents / Q000494:pharmacology; D000818:Animals; D017209:Apoptosis / Q000187:drug effects; D015153:Blotting, Western; D020169:Caspases / Q000378:metabolism; D016923:Cell Death / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects; D020552:Comet Assay; D004249:DNA Damage; D053938:DNA Fragmentation; D005455:Fluorescent Antibody Technique; D008297:Male; D008769:Methylnitronitrosoguanidine / Q000633:toxicity*; D051379:Mice; D008810:Mice, Inbred C57BL; D008928:Mitochondria / Q000187:drug effects / Q000378:metabolism; D009566:Nitrates / Q000378:metabolism*; D009569:Nitric Oxide / Q000378:metabolism; D030421:Peroxynitrous Acid / Q000378:metabolism; D011065:Poly(ADP-ribose) Polymerases / Q000378:metabolism*; D013481:Superoxides / Q000378:metabolism; D013601:T-Lymphocytes / Q000187:drug effects* / Q000201:enzymology / Q000378:metabolism*; D014443:Tyrosine / Q000378:metabolism*
D000477:Alkylating Agents; D009566:Nitrates; D013481:Superoxides; D008769:Methylnitronitrosoguanidine; D030421:Peroxynitrous Acid; D009569:Nitric Oxide; D014443:Tyrosine; D011065:Poly(ADP-ribose) Polymerases; D020169:Caspases