Carcinogenesis Advance Access originally published online on March 6, 2007
Carcinogenesis 2007 28(7):1430-1436; doi:10.1093/carcin/bgm029
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A modified host-cell reactivation assay to measure repair of alkylating DNA damage for assessing risk of lung adenocarcinoma
Department of Epidemiology, The University of Texas M. D. Anderson Cancer Center, 1515 Holcombe Boulevard, Houston, TX 77030, USA
* To whom correspondence should be addressed. Tel: +1 713 792 3020; Fax: +1 713 563 0999; Email: qwei{at}mdanderson.org
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.
Abbreviations: BPDE, benzo[a]pyrene diol epoxide; CI, confidence interval; DMS, dimethyl sulfate; DRC, DNA repair capacity; FBS, fetal bovine serum; HCR, host-cell reactivation; NER, nucleotide excision repair; NNK, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone; PHA, phytohemagglutinin
Received November 15, 2006; revised January 10, 2007; accepted February 5, 2007.