Modulation of DNA repair by various inhibitors of DNA synthesis following 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) induced DNA damage.
The tobacco specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is present in tobacco smoke and is hepatocarcinogenic in rats. Its bioactivation in rat hepatocytes leads to methylation and pyridyloxobutylation of DNA. Rat hepatocytes were cultured in serum-free William medium E on collagen-coated dishes. We demonstrated that some enzymes of the base and/or excision-repair pathways were involved in repair of NNK-induced DNA damage, measured by [methyl-3H] thymidine incorporation. Unscheduled DNA synthesis (UDS) induced by N-methyl-N-nitrosourea (MNU), NNK, N'-nitrosonornicotine (NNN) and 4-(acetoxymethylnitrosamino)-1-(3-pyridyl)-1-butanone (NNKOAc) increased 2.9-, 2.8-, 1.5- and 3.5-fold, respectively, suggesting that methylated and/or pyridyloxobutylated-DNA by these four nitroso compounds is repaired by the excision pathway. Moreover, levels of NNK-induced UDS were dose (1-3 mM) and time (1-18 h) dependent. Enzymes involved in the excision repair pathways were selectively inhibited. Inhibitors of DNA topoisomerase I (camptothecin) and topoisomerase II (etoposide, nalidixic acid) did not decrease the induction of UDS, suggesting that topoisomerases are not involved in the repair of NNK-induced damage. While aphidicolin and arabinocytidine (DNA polymerase alpha, delta, epsilon inhibitors) totally inhibited NNK- and NNKOAc-induced UDS, dideoxythymidine (DNA polymerase beta inhibitor) inhibited NNK- and NNKOAc-induced UDS by 40 and 33%, respectively. We conclude that DNA polymerase alpha, delta or epsilon and to a lesser degree polymerase beta are involved in the repair of pyridyloxobutylated DNA. Previous studies showed that inhibition of poly(ADP-ribosyl) polymerase (PARP) by 3-aminobenzamide (3-ab) facilitated DNA ligation. Our results demonstrate that 3-ab increased NNK-induced UDS, but does not affect NNKOAc-induced UDS. These observations suggest that the ligation step is rate limiting in the repair of methylated DNA but not of pyridyloxobutylated DNA.
Cloutier
J F
JF
Laboratory of Cancer Etiology and Chemoprevention, Faculty of Pharmacy, Laval University, Quebec City, Canada.
Castonguay
A
A
D000818:Animals; D002273:Carcinogens / Q000633:toxicity*; D002478:Cells, Cultured / Q000187:drug effects; D004249:DNA Damage; D019175:DNA Methylation; D004260:DNA Repair / Q000187:drug effects*; D008099:Liver / Q000187:drug effects*; D008114:Liver Neoplasms, Experimental / Q000139:chemically induced; D008297:Male; D008770:Methylnitrosourea / Q000633:toxicity; D009602:Nitrosamines / Q000633:toxicity*; D019384:Nucleic Acid Synthesis Inhibitors / Q000494:pharmacology*; D011725:Pyridines / Q000633:toxicity; D051381:Rats; D011916:Rats, Inbred F344; D014028:Tobacco Smoke Pollution / Q000009:adverse effects; D059004:Topoisomerase I Inhibitors
D002273:Carcinogens; D009602:Nitrosamines; D019384:Nucleic Acid Synthesis Inhibitors; D011725:Pyridines; D014028:Tobacco Smoke Pollution; D059004:Topoisomerase I Inhibitors; C081510:4-(acetoxymethylnitrosamino)-1-(3-pyridyl)-1-butanone; D008770:Methylnitrosourea; C016583:4-(N-methyl-N-nitrosamino)-1-(3-pyridyl)-1-butanone; C008655:N'-nitrosonornicotine