A Gas Chromatography/Electron Capture/Negative Chemical Ionization High-Resolution Mass Spectrometry Method for Analysis of Endogenous and Exogenous N7-(2-Hydroxyethyl)guanine in Rodents and Its Potential for Human Biological Monitoring

Abstract

A gas chromatography/electron capture/negative chemical ionization high-resolution mass spectrometry (GC/EC/NCI-HRMS) method was developed for quantitating N7-(2-hydroxyethyl)guanine (N7-HEG) with excellent sensitivity and specificity. [4,5,6,8-(13)C(4)]-N7-HEG was synthesized, characterized, and quantitated using HPLC/electrospray ionization mass spectrometry (HPLC/ESI-MS) so it could serve as an internal standard. After being converted to its corresponding xanthine and derivatized with pentafluorobenzyl (PFB) bromide twice, the PFB derivative of N7-HEG was characterized using GC/EC/NCI-HRMS carried out at full scan mode. The most abundant fragment was at m/z 555, with a molecular formula of C(21)H(9)N(4)O(3)F(10), resulting from the loss of one PFB group. By monitoring m/z 555.0515 (analyte) and m/z 559.0649 (internal standard), this assay demonstrated a linear relationship over a range of 1 fmol to 1 pmol of N7-HEG versus 20 fmol of [(13)C(4)]-N7-HEG on column. The limit of detection (LOD) for the complete assay was 600 amol (S/N = 5) injected on column. The variation of this assay was within 15% from 1 to 20 fmol of N7-HEG versus 2 fmol of [(13)C(4)]-N7-HEG with four replications for each calibration standard. Two hundred to three hundred micrograms of spleen DNA of control rats and mice and 100 microg of spleen DNA of rats and mice exposed to 3000 ppm ethylene for 6 h/day for 5 days were analyzed using GC/EC/NCI-HRMS. The amounts of N7-HEG varied from 0.2 to 0.3 pmol/micromol of guanine in tissues of control rats. Ethylene-exposed animals had 5-15-fold higher N7-HEG levels than controls. This assay was able to quantitate N7-HEG in 25-30 microg of DNA from human lymphocytes with excellent specificity. This was due in part to human tissues having 10-15-fold higher amounts of endogenous N7-HEG than rodents. These results show that this GC/EC/NCI-HRMS method is highly sensitive and specific and can be used in biological monitoring and molecular dosimetry and molecular epidemiology studies.

Dose-Response Relationships for N7-(2-Hydroxyethyl)Guanine Induced by Low-Dose [14C]Ethylene Oxide: Evidence for a Novel Mechanism of Endogenous Adduct Formation

“…Using our established LC-MS/MS assay for N7-HEG, it was possible to accurately quantify the background (unlabeled) adduct level in rat tissues, distinct from those formed as a consequence of [ ]N7-HEG formation in rat tissues was around 10 to 20 adducts/10 9 nucleotides; this is 5-to 100-fold lower than the endogenous level of these adducts reported in WBCs isolated from unexposed humans (16)(17)(18).…”

“…A confounding factor in evaluating the risks associated with EO inhalation is the fact that ethylene is also generated in vivo during normal physiologic processes and can be converted to the epoxide by cytochrome P450 2E1 (15). Humans are therefore continually exposed to EO, as illustrated by detectable N7-HEG at levels of f1 to 10/10 7 nucleotides in lymphocytes isolated from people not knowingly in contact with EO (16)(17)(18). Physiologic sources of ethylene are believed to include methionine oxidation, lipid peroxidation, and the metabolizing activity of intestinal bacteria (19)(20)(21); however, the mechanisms of formation in mammalian systems have not been defined in any of these cases and the origins of endogenous N7-HEG adducts have never been directly shown.…”

DNA adducts in granulocytes of hospital workers exposed to ethylene oxide

“…One of the limitations of this study is the small sample size with only five never smokers and one current smoker in the control group. Based on quantification methods with varying degrees of sensitivity and specificity [Fost et al, 1989;Bolt et al, 1997;Wu et al, 1999;Zhao et al, 1999;Zhao and Hemminki, 2002], the background levels of N7-HEG adducts in various tissues of nonsmokers and smokers not occupationally exposed to EtO appear to be mostly less than 10 adducts/10 7 nucleotides.…”

“…N7-(2 0 -hydroxyethyl)guanine (N7-HEG), which represents about 90% of the alkylated sites, is the major DNA adduct induced in vitro [Segerback, 1990;Li et al, 1992] and in experimental animals [Segerback, 1983;Walker et al, 1993;Wu et al, 1999;van Sittert et al, 2000]. Although N7-HEG adducts are not considered directly promutagenic, they are measurable, and thus could serve as a surrogate biomarker for other minor and more promutagenic DNA adducts of EtO [van Sittert et al, 2000].…”

Analysis of 7‐methylguanine using isotope dilution and gas chromatography/electron‐capture negative chemical ionization mass spectrometry

“…These calibration solutions were derivatized with PFBBr by modifying the procedure previously described. 9 Briefly, samples were cooled in ice and reacted with tert-butyl nitrite in 6 M HCl (degassed with N 2 ) at 48C for 4 h and converted into 7-methylxanthine. The samples were then evaporated in vacuo, and subjected to liquid-liquid extraction using HPLC water (50 mL) and ethyl acetate (150 mL).…”