TOXI 136 |
| Considerable evidence in the literature demonstrates the exposure of humans to an unknown ethylating agent. Previous studies have demonstrated the presence of 7-ethylguanine and 3-ethyladenine in urine, O4-ethylthymidine in human lung, and ethylvaline in hemoglobin. Some studies also show higher levels of ethyl adducts in smokers than in non-smokers and there is convincing evidence for an uncharacterized ethylating agent in cigarette smoke. To further investigate this question, we have developed a method for analysis of 7-ethylguanine in human liver DNA. There are no previous studies on this adduct in liver. [15N5]7-Ethylguanine was synthesized and used as an internal standard. DNA was isolated from human liver, internal standard was added, and the mixture was heated at 100°C for 1h to release 7-ethylguanine. High molecular weight material was removed by filtration and the filtrate was applied to a Strata X solid phase extraction cartridge and eluted with 70% methanol. The eluant was analyzed by liquid chromatography-electrospray ionization-tandem mass spectrometry with selected reaction monitoring at m/z 180 (M+H)+ → m/z 152 for 7-ethylguanine and m/z 185 → m/z 157 for the internal standard. Accuracy and precision were determined by adding 7-ethylguanine to rat liver DNA: accuracy, R2=0.99; precision, coefficient of variation=9%. The limit of detection was 0.25 fmol 7-ethylguanine on column (S/N=4) and the recovery was 75%. Analysis of human liver DNA, mean ± S.D., 0.77 ± 0.40 mg, demonstrated that 25 of 26 samples were positive for 7-ethylguanine, mean ± S.D., 42.2 ± 43.0 (range ND–174) fmol/μmol guanine. These results demonstrate that 7-ethylguanine is a common DNA adduct in human liver. Levels were approximately one tenth of those of N2-ethylidene-dG and about ten times greater than those of the crotonaldehyde derived- 1,N2-propano-dG adducts in liver. Thus, 7-ethylguanine in human liver and possibly other tissues has likely endogenous sources that require further investigation. |
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General Papers
1:00 PM-4:45 PM, Wednesday, August 22, 2007 BCEC -- 258C, Oral
Division of Chemical Toxicology |