Quantitative determination of urinary N7-ethylguanine in smokers and non-smokers using an isotope dilution liquid chromatography/tandem mass spectrometry with on-line analyte enrichment.
Previous studies demonstrated the presence of unknown direct-acting ethylating agents arising from cigarette smoke. We hypothesized that such agents would also lead to ethylation of guanine in DNA followed by depurination/repair and excretion of N7-ethylguanine (N7-EtG) in urine. In this study, a highly specific and sensitive liquid chromatographic/tandem mass spectrometric (LC/MS/MS) method was firstly developed for measuring urinary N7-EtG. With the use of an isotope internal standard (15N5-N7-EtG) and on-line enrichment techniques, the detection limit of this method was estimated as 0.59 pg/ml (0.33 pmol) on-column. This method was then applied to measure urinary samples obtained from 35 non-smokers and 32 smokers with dietary control. The results showed that the mean urinary levels of N7-EtG were 85.5+/-105 and 28.1+/-19.4 pg/mg creatinine for smokers and non-smokers, respectively. Smokers had about three times higher level of N7-EtG than non-smokers (P<0.005). It was further noted that the urinary level of N7-EtG was significantly associated with cotinine for smokers (r=0.49, P<0.005). Taken together, this is the first study that demonstrated the presence of N7-EtG in urine, and that cigarette smoke was highly responsible for the increased urinary excretion of N7-EtG. This non-invasive measurement of urinary N7-EtG would be useful for the surveillance of ethylating agent exposure and its associated cancer risk in the future.
Chao
Mu-Rong
MR
Department of Occupational Safety and Health, Chung Shan Medical University, No. 110, Sec.1, Chien-Kuo N Road, Taichung 402, Taiwan.
Wang
Chien-Jen
CJ
Chang
Louis W
LW
Hu
Chiung-Wen
CW
D000477:Alkylating Agents / Q000652:urine*; D015415:Biomarkers / Q000652:urine; D002853:Chromatography, Liquid; D003404:Creatinine / Q000652:urine; D004247:DNA / Q000737:chemistry; D004249:DNA Damage; D004032:Diet; D006147:Guanine / Q000031:analogs & derivatives* / Q000652:urine; D006801:Humans; D008297:Male; D009587:Nitrogen Isotopes; D009862:Online Systems; D011865:Radioisotope Dilution Technique; D012907:Smoking; D021241:Spectrometry, Mass, Electrospray Ionization
D000477:Alkylating Agents; D015415:Biomarkers; D009587:Nitrogen Isotopes; C022007:7-ethylguanine; D006147:Guanine; D004247:DNA; D003404:Creatinine