The purposes of the present study were: (i) to investigate the potential use of several biomarkers as quantitative indicators of the in vivo conversion of ethylene (ET) to ethylene oxide (EO); (ii) to produce molecular dosimetry data that might improve assessment of human risk from exogenous ET exposures. Groups (n = 7/group) of male F344 rats and B6C3F1 mice were exposed by inhalation to 0 and 3000 p. p.m. ET for 1, 2 or 4 weeks (6 h/day, 5 days/week) or to 0, 40, 1000 and 3000 p.p.m. ET for 4 weeks. N:-(2-hydroxyethyl)valine (HEV), N:7-(2-hydroxyethyl) guanine (N7-HEG) and HPRT: mutant frequencies were assessed as potential biomarkers for determining the molecular dose of EO resulting from exogenous ET exposures of rats and mice, compared with background biomarker values. N7-HEG was quantified by gas chromatography coupled with high resolution mass spectrometry (GC-HRMS), HEV was determined by Edman degradation and GC-HRMS and HPRT: mutant frequencies were measured by the T cell cloning assay. N7-HEG accumulated in DNA with repeated exposure of rodents to 3000 p.p.m. ET, reaching steady-state concentrations around 1 week of exposure in most tissues evaluated (brain, liver, lung and spleen). The dose-response curves for N7-HEG and HEV were supralinear in exposed rats and mice, indicating that metabolic activation of ET was saturated at exposures >/=1000 p.p.m. ET. Exposures of mice and rats to 200 p.p.m. EO for 4 weeks (as positive treatment controls) led to significant increases in HPRT: mutant frequencies over background in splenic T cells from exposed rats and mice, however, no significant mutagenic response was observed in the HPRT: gene of ET-exposed animals. Comparisons between the biomarker data for both unexposed and ET-exposed animals, the dose-response curves for the same biomarkers in EO-exposed rats and mice and the results of the rodent carcinogenicity studies of ET and EO suggest that too little EO arises from exogenous ET exposure to produce a significant mutagenic response or a carcinogenic response under standard bioassay conditions.

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.

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The need for specificity and sensitivity in the analysis of DNA adducts has led the development of GC/MS methods. Such methods require chemical derivatization (i.e. silylation, electrophore labelling), which can also bring its own sets of problems, including the production of artifacts, interferences and sample to sample variability in derivatization. To obviate such problems, a liquid chromatographic/electrospray ionization mass spectrometric (LC/ESI-MS) method was developed to quantify N2,3-ethenoguanine (epsilon Gua), a promutagenic DNA adduct of vinyl chloride exposure. The response of epsilon Gua to isotopically labelled internal standard [13C4]epsilon Gua was linear (r2 = 0.999) and reproducible from 0.027 to 0.538 pmol microliter-1. We obtained an accuracy of 86 +/- 14% by analyzing chloroethylene oxide (CEO)-treated calf thymus DNA enriched with authentic epsilon Gua. The analysis of CEO-treated calf thymus DNA samples not enriched with authentic epsilon Gua provided a precision of 15%. The detection limits with a signal-to-noise ratio (S/N) 2.5:1 were obtained in the determination of authentic epsilon Gua at 5 fmol per injection. The detection limit obtained in the routine analysis of the biological samples was 50 fmol epsilon Gua with S/N = 3:1. The applicability of the method was established by determining epsilon Gua in rats treated with CEO by portal vein injection and an unexposed human liver. It was observed that the concentration of epsilon Gua in the rat livers increased with increase in dose and was inversely related to the time after, CEO exposure. This trend suggests rapid repair of the adduct in rat livers. In the human liver DNA sample, epsilon Gua was quantitated at 0.06 +/- 0.01 pmol mg-1 DNA.

The analytical potential of gas chromatography/electron capture negative chemical ionization high-resolution mass spectrometry (HRMS) for characterization and quantitation of DNA and hemoglobin adducts was demonstrated using three model compounds: N2, 3-ethenoguanine (EG), 7-(2-hydroxyethyl)guanine (7-HEG), and N-(2-hydroxyethyl)valine (HEV). At a resolving power of 10 000, the signal-to-noise (S/N) ratios obtained from quantitative selected ion monitoring (SIM) experiments using biological samples were comparable to or better than existing unit mass resolution experiments due to the reduction of chemical noise from the use of narrower mass windows. The specificity gained by HRMS was essential for quantitation of ultratrace amounts near the limit of detection since coeluting interferences of the analyte or internal standard can lead to inaccurate measurement of response factors. The limit of detection (LOD) was 100 amol (S/N = 5) using a pure standard of TTB2-EG. The LOD for complete assays using spiked samples was 500 amol (S/N = 5) for EG and 600 amol (S/N = 5) injected for 7-HEG. The standard deviation (SD) for the HRMS quantitative measurements was typically less than 10%. The SD for the complete biological assays as determined by spiking replicate samples was less than 15%. This method has adequate sensitivity and specificity to accurately measure DNA and protein adducts as low as endogenous concentrations in rodent and human tissues.