title: A modified host-cell reactivation assay to measure repair of alkylating DNA damage for assessing risk of lung adenocarcinoma.
abstract: The nicotine-derived nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) induces lung adenocarcinoma through formation of DNA adducts. Our previous research on susceptibility to tobacco-induced carcinogenesis focused on benzo[a]pyrene diol epoxide (BPDE) as the in vitro mutagen for phenotype measurements of DNA repair capacity (DRC) in mammalian cells. Here, we present a modified host-cell reactivation (HCR) assay to measure lymphocytic DRC for alkylating DNA damage as is induced by the tobacco-specific nitrosamine, NNK. We substituted dimethyl sulfate (DMS) to create alkylating damage in pCMVluc plasmid DNA and established the damage-repair dose-response curves in both normal and nucleotide excision repair-deficient lymphoblastoid cell lines and in phytohemagglutinin (PHA)-stimulated primary lymphocytes. We then successfully measured the DRC in PHA-stimulated lymphocytes from 48 patients with lung adenocarcinoma and 45 cancer-free controls and tested our hypothesis that lower DRC for alkylating damage is associated with an increased risk of lung adenocarcinoma. The cases exhibited a lower mean DRC than did the controls. A >3-fold increased risk (odds ratio = 3.21; 95% confidence interval = 1.25-8.21) was found for those with DRC levels below the control median. There was no correlation between the DRC measured with this DMS-HCR assay and that from the parallel BPDE-HCR assay. Interestingly, risk increased to >10-fold for those with sub-optimal DRC measured by both DMS- and BPDE-HCR assays. We conclude that variability in DRC is a risk factor for lung cancer and our results provide proof of principle for a new assay that can assess DRC for NNK-induced DNA damage.
authors.affiliation: Department of Epidemiology, The University of Texas M. D. Anderson Cancer Center, 1515 Holcombe Boulevard, Houston, TX 77030, USA.
authors.forename: Luo
authors.initials: L
authors.lastname: Wang
authors.forename: Qingyi
authors.initials: Q
authors.lastname: Wei
authors.forename: Qiuling
authors.initials: Q
authors.lastname: Shi
authors.forename: Zhaosheng
authors.initials: Z
authors.lastname: Guo
authors.forename: Yawei
authors.initials: Y
authors.lastname: Qiao
authors.forename: Margaret R
authors.initials: MR
authors.lastname: Spitz
mesh_terms: D015123:7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide / Q000633:toxicity; D000230:Adenocarcinoma / Q000097:blood / Q000378:metabolism*; D000328:Adult; D000368:Aged; D000477:Alkylating Agents / Q000633:toxicity*; D000478:Alkylation; D001681:Biological Assay; D002273:Carcinogens / Q000633:toxicity*; D016022:Case-Control Studies; D002478:Cells, Cultured; D004249:DNA Damage; D004260:DNA Repair; D005260:Female; D006801:Humans; D066298:In Vitro Techniques; D008175:Lung Neoplasms / Q000097:blood / Q000378:metabolism*; D008213:Lymphocyte Activation; D008214:Lymphocytes / Q000378:metabolism*; D008297:Male; D008875:Middle Aged; D009602:Nitrosamines / Q000633:toxicity; D010835:Phytohemagglutinins / Q000494:pharmacology; D010865:Pilot Projects; D010957:Plasmids; D012307:Risk Factors; D013463:Sulfuric Acid Esters / Q000633:toxicity
chemical_list: D000477:Alkylating Agents; D002273:Carcinogens; D009602:Nitrosamines; D010835:Phytohemagglutinins; D013463:Sulfuric Acid Esters; D015123:7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide; C016583:4-(N-methyl-N-nitrosamino)-1-(3-pyridyl)-1-butanone; C007482:dimethyl sulfate