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.
Department of Occupational Safety and Health, Chung Shan Medical University, No. 110, Sec.1, Chien-Kuo N Road, Taichung 402, Taiwan.
Mu-Rong
MR
Chao
Chien-Jen
CJ
Wang
Louis W
LW
Chang
Chiung-Wen
CW
Hu
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