Determination of Endogenous and Exogenously Derived N7-(2-Hydroxyethyl)guanine Adducts in Ethylene Oxide-Treated Rats

Abstract

Ethylene oxide (EO) is one of the most widely used intermediates in the chemical industry. It is also formed endogenously as a result of cytochrome P450-mediated metabolism of ethylene, which is ubiquitous in the environment. Additionally, ethylene is generated in vivo during normal physiological processes such as methionine oxidation and lipid peroxidation; therefore, humans are continually exposed to EO. EO is classed by the IARC as carcinogenic to humans and reacts with DNA, primarily forming N7-(2-hydroxyethyl)guanine adducts (N7-HEG), which can be used as biomarkers of exposure and potential cancer risk. To assess the risks to humans associated with occupational exposure to low EO concentrations, it is necessary to establish the relative contribution of DNA damage arising from endogenous and exogenously derived EO. Using a newly developed highly sensitive LC-MS/MS assay with selected reaction monitoring that offers a limit of detection of 0.1 fmol of N7-HEG on column, we have established background levels of N7-HEG (1.1-3.5 adducts/10(8) nucleotides) in tissues of rats. Following intraperitoneal administration of a single dose or three daily doses of EO (0.01-1.0 mg/kg), N7-HEG adducts generally increased with dose, except at the lowest concentration where total N7-HEG levels were no different to that detected in control animals, indicating that any increase was negligible as compared to the endogenous damage already present. In the 3 day study, the kinetics of adduct removal were also investigated and in comparing N7-HEG formation in the two studies, DNA damage did not appear to accumulate with repeated administration.

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

“…Unlabeled N7-HEG adducts were quantified using our validated LC-MS/MS method as described previously (26). Each sample of hydrolyzed DNA (50 Ag) was injected onto a Zorbax 300SB C 18 column (5 Am, 4.6 Â 250 mm; Agilent), attached to a KrudKatcher disposable precolumn (0.5 Am) filter (Phenomenex), and eluted using a gradient mobile phase of (A) 0.5% acetic acid and (B) 100% methanol as follows: 95% A from 0 to 7 min, 95% to 70% A from 7 to 9 min, and then 70% to 95% A from 9 to 11 min with a flow rate of 0.85 mL/min.…”

“…Each sample of hydrolyzed DNA (50 Ag) was injected onto a Zorbax 300SB C 18 column (5 Am, 4.6 Â 250 mm; Agilent), attached to a KrudKatcher disposable precolumn (0.5 Am) filter (Phenomenex), and eluted using a gradient mobile phase of (A) 0.5% acetic acid and (B) 100% methanol as follows: 95% A from 0 to 7 min, 95% to 70% A from 7 to 9 min, and then 70% to 95% A from 9 to 11 min with a flow rate of 0.85 mL/min. Samples were analyzed in the positive ionization mode using selected reaction monitoring (SRM) for the transitions m/z 196!152 and 201!157 (26). Adduct values were corrected to take into account any contribution from the unlabeled fraction of the [ 14 C]N7-HEG peak of interest.…”

Use of DNA adducts to identify human health risk from exposure to hazardous environmental pollutants: The increasing role of mass spectrometry in assessing biologically effective doses of genotoxic carcinogens

“…adducts/10 8 nucleotides [47]. Thermal depurination of DNA was also recently reported to have been used by Lao et al [43] to study pyridyloxobutyl adducted guanine, as a possible marker of tobacco specific nitrosamine exposure.…”

The application of mass spectrometry in molecular dosimetry: Ethylene oxide as an example

“…Isotope-dilution LC/MS/MS methods have been developed to analyze N7-HEG in tissue DNA and human urine samples (Koc & Swenberg, 2002;Rusyn et al, 2005;Marsden et al, 2007;Huang et al, 2008). Internal standard ( 13 C 4 -labeled N7-HEG) was spiked into DNA solution at the beginning of sample preparation.…”

“…Another istope-dilution method was also developed to quantify N7-HEG in tissues of control and EO-treated rodents with LC/MS/MS (Marsden et al, 2007).…”