Detection of DNA damage derived from a direct acting alkylating agent present in cigarette smoke using liquid chromatography tandem mass spectrometry
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Slide 1 :
Detection of DNA damage derived from a direct acting alkylating agent present in cigarette smoke using liquid chromatography tandem mass spectrometry Raj Singh Cancer Biomarkers and Prevention Group Biocentre University of Leicester
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Outline of talk Smoking and carcinogenesis The biology and chemistry of alkylating aganets Development of an LC-MS/MS method Results
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Smoking cigarettes is associated with the development of numerous human cancers and accounts for 30% of all deaths related to cancers in more developed countries. Lung cancer is the most prevalent cancer arising from cigarette smoking with approximately 87% of all lung cancer cases being caused by exposure to cigarette smoke. Cigarette smoking and carcinogenesis
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In total cigarette smoke contains about 4000 chemicals of which about 60 are classified carcinogens in humans and animals as assessed by the International Agency for Research on Cancer (IARC). Cigarette smoking and carcinogenesis A significant number of epidemiological and clinical studies have reported the association of increased DNA adduct levels with occurrence and prevalence of cigarette related cancers such as lung, head and neck and bladder.
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Cigarette smoke and carcinogenesis Some carcinogens present in tobacco smoke: Benzo(a)pyrene 5-methylchrysene N-nitrosodimethylamine (NDMA) N-nitrosodiethylamine (NDEA) 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) Ethylene oxide 1,3-butadiene Acetaldehyde Crotonaldehyde 4-aminobiphenyl Vinyl chloride Free Radicals The majority of chemicals present in cigarette smoke require cytochrome P450 dependent metabolism to form electrophilic species that covalently react with DNA to form adducts. There are a few chemicals such as ethylene oxide and acetaldehyde, which do not require metabolism for reaction with the DNA..
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p53 mutations in smoking-associated cancers p53 mutations found in smoking related lung cancers. G to T transversions correspond to formation of PAH and nitrosoamine derived adducts. Denissenko, M.F. et al. (1996) Science 274, 430-432.
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Presence of a direct acting alkylating agent in cigarette smoke? A further study assessed exposure of smokers to ethylating agents in cigarette smoke by determining the N-terminal N-ethylvaline in haemoglobin, which was shown to be significantly elevated compared to non smokers. Carmella, S.G. et al. (2002) Carcinogenesis 23, 1903-1910. Evidence suggests that there is a direct acting ethylating agent present in cigarette smoke of unknown chemical identity. A human study showed that there were higher levels N-3 ethyladenine in the urine of smokers compared to that of the non smokers. Prevost, V. and Shuker, D.E.G (1996) Chem. Res. Toxicol. 9, 439-444. The levels of N-nitrosamines in cigarette smoke such as N-nitrosodiethylamine and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) range from 0 to 2.8 ng and 80-770 ng per cigarette, respectively. In contrast aldehydes and volatile compounds like benzene and butadiene are found levels ranging from 10-1000 µg per cigarette.
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The structures of simple alkylating agents
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Cyt. P450 Activation of N-nitrosodiethylamine
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The different sites of modification in DNA by alkylating agents.
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Percentage of total methylation or ethylation of DNA in vitro and in vivo. ( ) indicates in vivo data nd indicates adduct not detected or below limits of detection - indicates data not reported It is generally considered that the more reactive the alkylating agent eg. MNU or ENU the more likely it is to form oxygen adducts via a SN1 mechanism than less reactive agents eg. DMS or DES which react via SN2 mechanism
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O6-alkylguanine-DNA Alkyltransferase (AGT) The presence of a O6 alkylguanine adduct results in the direct stoichiometric transfer of the alkyl group from the DNA to a cysteine group present in the active site of the alkyltransferase forming S-alkylcysteine. O4 alkylthymine is less efficiently repaired Repair of alkylated DNA damage
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Chemical properties of alkyl DNA adducts
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N-7EtG as a biomarker of alkylated DNA damage The N-7EtG DNA adduct represents a good biomarker for determining exposure to ethylating agents since it is the major adduct formed by alkylating agents and is slowly repaired. Not regarded to be promutagenic Though depurination may occur resulting in abasic sites and GC to TA mutations following replication. These apurinic sites are repaired by base excision repair pathway.
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Different methods used to detect methyl and ethyl adducts in DNA samples
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Procedure for the determination of N-7EtG in DNA
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Multiple Reaction Monitoring (MRM)
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Plan of experiment – DES + CT DNA 8h incubation at Room Temp.
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Levels of N-7EtG in DES exposed CT DNA samples
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Plan of experiment – Cigarette smoke + CT DNA 48h incubation at 37oC
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Diagram of apparatus used for trapping cigarette smoke
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Conclusions Sensitive LC-MS/MS method developed for the detection of N-7EtG – as sensitive as 32P-postlabelling. LOD = 2fmol (0.6 N-7EtG per 108 nucleotides) Confirmed the presence of a direct acting alkylating agent in cigarette smoke – increased N-7EtG levels with number of cigarettes. Concentration of DES required to form levels of N-7EtG observed following cigarette smoke exposure.
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